Calcium dependent current recordings in Xenopus laevis oocytes in microgravity

NASA Astrophysics Data System (ADS)

Wuest, Simon L.; Roesch, Christian; Ille, Fabian; Egli, Marcel

2017-12-01

Mechanical unloading by microgravity (or weightlessness) conditions triggers profound adaptation processes at the cellular and organ levels. Among other mechanisms, mechanosensitive ion channels are thought to play a key role in allowing cells to transduce mechanical forces. Previous experiments performed under microgravity have shown that gravity affects the gating properties of ion channels. Here, a method is described to record a calcium-dependent current in native Xenopus laevis oocytes under microgravity conditions during a parabolic flight. A 3-voltage-step protocol was applied to provoke a calcium-dependent current. This current increased with extracellular calcium concentration and could be reduced by applying extracellular gadolinium. The custom-made ;OoClamp; hardware was validated by comparing the results of the 3-voltage-step protocol to results obtained with a well-established two-electrode voltage clamp (TEVC). In the context of the 2nd Swiss Parabolic Flight Campaign, we tested the OoClamp and the method. The setup and experiment protocol worked well in parabolic flight. A tendency that the calcium-dependent current was smaller under microgravity than under 1 g condition could be observed. However, a conclusive statement was not possible due to the small size of the data base that could be gathered.

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.

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 Mechanism of Extracellular Stimulation of Nerve Cells on an Electrolyte-Oxide-Semiconductor Capacitor

PubMed Central

Schoen, Ingmar; Fromherz, Peter

2007-01-01

Extracellular excitation of neurons is applied in studies of cultured networks and brain tissue, as well as in neuroprosthetics. We elucidate its mechanism in an electrophysiological approach by comparing voltage-clamp and current-clamp recordings of individual neurons on an insulated planar electrode. Noninvasive stimulation of neurons from pedal ganglia of Lymnaea stagnalis is achieved by defined voltage ramps applied to an electrolyte/HfO2/silicon capacitor. Effects on the smaller attached cell membrane and the larger free membrane are distinguished in a two-domain-stimulation model. Under current-clamp, we study the polarization that is induced for closed ion channels. Under voltage-clamp, we determine the capacitive gating of ion channels in the attached membrane by falling voltage ramps and for comparison also the gating of all channels by conventional variation of the intracellular voltage. Neuronal excitation is elicited under current-clamp by two mechanisms: Rising voltage ramps depolarize the free membrane such that an action potential is triggered. Falling voltage ramps depolarize the attached membrane such that local ion currents are activated that depolarize the free membrane and trigger an action potential. The electrophysiological analysis of extracellular stimulation in the simple model system is a basis for its systematic optimization in neuronal networks and brain tissue. PMID:17098803

Nav1.7-A1632G Mutation from a Family with Inherited Erythromelalgia: Enhanced Firing of Dorsal Root Ganglia Neurons Evoked by Thermal Stimuli.

PubMed

Yang, Yang; Huang, Jianying; Mis, Malgorzata A; Estacion, Mark; Macala, Lawrence; Shah, Palak; Schulman, Betsy R; Horton, Daniel B; Dib-Hajj, Sulayman D; Waxman, Stephen G

2016-07-13

Voltage-gated sodium channel Nav1.7 is a central player in human pain. Mutations in Nav1.7 produce several pain syndromes, including inherited erythromelalgia (IEM), a disorder in which gain-of-function mutations render dorsal root ganglia (DRG) neurons hyperexcitable. Although patients with IEM suffer from episodes of intense burning pain triggered by warmth, the effects of increased temperature on DRG neurons expressing mutant Nav1.7 channels have not been well documented. Here, using structural modeling, voltage-clamp, current-clamp, and multielectrode array recordings, we have studied a newly identified Nav1.7 mutation, Ala1632Gly, from a multigeneration family with IEM. Structural modeling suggests that Ala1632 is a molecular hinge and that the Ala1632Gly mutation may affect channel gating. Voltage-clamp recordings revealed that the Nav1.7-A1632G mutation hyperpolarizes activation and depolarizes fast-inactivation, both gain-of-function attributes at the channel level. Whole-cell current-clamp recordings demonstrated increased spontaneous firing, lower current threshold, and enhanced evoked firing in rat DRG neurons expressing Nav1.7-A1632G mutant channels. Multielectrode array recordings further revealed that intact rat DRG neurons expressing Nav1.7-A1632G mutant channels are more active than those expressing Nav1.7 WT channels. We also showed that physiologically relevant thermal stimuli markedly increase the mean firing frequencies and the number of active rat DRG neurons expressing Nav1.7-A1632G mutant channels, whereas the same thermal stimuli only increase these parameters slightly in rat DRG neurons expressing Nav1.7 WT channels. The response of DRG neurons expressing Nav1.7-A1632G mutant channels upon increase in temperature suggests a cellular basis for warmth-triggered pain in IEM. Inherited erythromelalgia (IEM), a severe pain syndrome characterized by episodes of intense burning pain triggered by warmth, is caused by mutations in sodium channel Nav1.7, which are preferentially expressed in sensory and sympathetic neurons. More than 20 gain-of-function Nav1.7 mutations have been identified from IEM patients, but the question of how warmth triggers episodes of pain in IEM has not been well addressed. Combining multielectrode array, voltage-clamp, and current-clamp recordings, we assessed a newly identified IEM mutation (Nav1.7-A1632G) from a multigeneration family. Our data demonstrate gain-of-function attributes at the channel level and differential effects of physiologically relevant thermal stimuli on the excitability of DRG neurons expressing mutant and WT Nav1.7 channels, suggesting a cellular mechanism for warmth-triggered pain episodes in IEM patients. Copyright © 2016 the authors 0270-6474/16/367512-12$15.00/0.

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.

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.

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.

Planar patch clamp for neuronal networks--considerations and future perspectives.

PubMed

Bosca, Alessandro; Martina, Marzia; Py, Christophe

2014-01-01

The patch-clamp technique is generally accepted as the gold standard for studying ion channel activity allowing investigators to either "clamp" membrane voltage and directly measure transmembrane currents through ion channels, or to passively monitor spontaneously occurring intracellular voltage oscillations. However, this resulting high information content comes at a price. The technique is labor-intensive and requires highly trained personnel and expensive equipment. This seriously limits its application as an interrogation tool for drug development. Patch-clamp chips have been developed in the last decade to overcome the tedious manipulations associated with the use of glass pipettes in conventional patch-clamp experiments. In this chapter, we describe some of the main materials and fabrication protocols that have been developed to date for the production of patch-clamp chips. We also present the concept of a patch-clamp chip array providing high resolution patch-clamp recordings from individual cells at multiple sites in a network of communicating neurons. On this chip, the neurons are aligned with the aperture-probes using chemical patterning. In the discussion we review the potential use of this technology for pharmaceutical assays, neuronal physiology and synaptic plasticity studies.

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.

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.

Membrane currents underlying activity in frog sinus venosus

PubMed Central

Brown, Hilary F.; Giles, Wayne; Noble, Susan J.

1977-01-01

1. The spontaneous electrical activity of small strips of muscle from the sinus venosus region of the heart of Rana catesbeiana was investigated using the double sucrose gap technique. The voltage clamp was used to record the ionic currents underlying the pace-maker depolarization and the action potential. 2. The records of spontaneous electrical activity are very similar to those obtained from the sinus venosus using micro-electrodes. Moreover, the pace-maker activity is almost completely insensitive to tetrodotoxin (TTX) at 2·0 × 10-6 g/ml., which suggests that the pace-maker responses can be classified as primary, as opposed to follower pacing. 3. In response to short rectangular depolarizing voltage clamp pulses, only one inward current is activated. This current is almost completely insensitive to TTX but can be blocked by manganese ions. It appears, therefore, to be equivalent to the slow inward (Ca2+/Na+) current, Isi, of other cardiac tissues. The threshold for Isi is near to the maximum diastolic potential, indicating that it must be activated during the pace-maker depolarization. 4. Interruption of the normal pace-maker depolarization by rapid activation of the voltage clamp circuit reveals the time-dependent decay of outward current. This current reverses between -75 and -90 mV and, therefore, is probably carried mainly by potassium ions. 5. Outward current decay is not a simple exponential, and Hodgkin—Huxley analysis suggests that two distinct components of outward current may be present. One of these is activated in the potential range of the pace-maker depolarization and the other at more positive potentials. Both outward currents reach full, steady-state activation at about zero mV, i.e. within the `plateau' range of the sinus action potential. 6. These results are compared with other recently published voltage clamp data from the rabbit sino—atrial node. 7. A hypothesis for the generation of pace-maker activity is presented which involves (i) decay of outward current and (ii) activation of the slow inward current, Isi. PMID:303699

Methods for stable recording of short-circuit current in a Na+-transporting epithelium.

PubMed

Gondzik, Veronika; Awayda, Mouhamed S

2011-07-01

Epithelial Na(+) transport as measured by a variety of techniques, including the short-circuit current technique, has been described to exhibit a "rundown" phenomenon. This phenomenon manifests as time-dependent decrease of current and resistance and precludes the ability to carry out prolonged experiments aimed at examining the regulation of this transport. We developed methods for prolonged stable recordings of epithelial Na(+) transport using modifications of the short-circuit current technique and commercial Ussing-type chambers. We utilize the polarized MDCK cell line expressing the epithelial Na(+) channel (ENaC) to describe these methods. Briefly, existing commercial chambers were modified to allow continuous flow of Ringer solution and precise control of such flow. Chamber manifolds and associated plumbing were modified to allow precise temperature clamp preventing temperature oscillations. Recording electrodes were modified to eliminate the use of KCl and prevent membrane depolarization from KCl leakage. Solutions utilized standard bicarbonate-based buffers, but all gasses were prehydrated to clamp buffer osmolarity. We demonstrate that these modifications result in measurements of current and resistance that are stable for at least 2 h. We further demonstrate that drifts in osmolarity similar to those obtained before prior to our modifications can lead to a decrease of current and resistance similar to those attributed to rundown.

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.

Decrement of GABAA receptor-mediated inhibitory postsynaptic currents in dentate granule cells in epileptic hippocampus.

PubMed

Isokawa, M

1996-05-01

1. Inhibitory postsynaptic currents (IPSCs) were studied in hippocampal dentate granule cells (DGCs) in the pilocarpine model and human temporal lobe epilepsy, with the use of the whole cell patch-clamp recording technique in slice preparations. 2. In the pilocarpine model, hippocampal slices were prepared from rats that were allowed to experience spontaneous seizures for 2 mo. Human hippocampal specimens were obtained from epileptic patients who underwent surgical treatment for medically intractable seizures. 3. IPSCs were generated by single perforant path stimulation and recorded at a membrane potential (Vm) of 0 mV near the reversal potential of glutamate excitatory postsynaptic currents in the voltage-clamp recording. IPSCs were pharmacologically identified as gamma-aminobutyric acid-A (GABAA) IPSCs by 10 microM bicuculline methiodide. 4. During low-frequency stimulation, IPSCs were not different in amplitude among non-seizure-experienced rat hippocampi, human nonsclerotic hippocampi, seizure-experienced rat hippocampi, and human sclerotic hippocampi. In the last two groups of DGCs, current-clamp recordings indicated the presence of prolonged excitatory postsynaptic potentials (EPSPs) mediated by the N-methyl-D-aspartate (NMDA) receptor. 5. High-frequency stimulation, administered at Vm = -30 mV to activate NMDA currents, reduced GABAA IPSC amplitude specifically in seizure-experienced rat hippocampi (t = 2.5, P < 0.03) and human sclerotic hippocampi (t = 7.7, P < 0.01). This reduction was blocked by an NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV) (50 microM). The time for GABAA IPSCs to recover to their original amplitude was also shortened by the application of APV. 6. I conclude that, when intensively activated, NMDA receptor-mediated excitatory transmission may interact with GABAergic synaptic inhibition in DGCs in seizure-experienced hippocampus to transiently reduce GABA(A) receptor-channel function. Such interactions may contribute to give rise to epileptic excitation in chronically seizure-prone hippocampus.

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

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.

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

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.

Effect of electrical coupling on ionic current and synaptic potential measurements.

PubMed

Rabbah, Pascale; Golowasch, Jorge; Nadim, Farzan

2005-07-01

Recent studies have found electrical coupling to be more ubiquitous than previously thought, and coupling through gap junctions is known to play a crucial role in neuronal function and network output. In particular, current spread through gap junctions may affect the activation of voltage-dependent conductances as well as chemical synaptic release. Using voltage-clamp recordings of two strongly electrically coupled neurons of the lobster stomatogastric ganglion and conductance-based models of these neurons, we identified effects of electrical coupling on the measurement of leak and voltage-gated outward currents, as well as synaptic potentials. Experimental measurements showed that both leak and voltage-gated outward currents are recruited by gap junctions from neurons coupled to the clamped cell. Nevertheless, in spite of the strong coupling between these neurons, the errors made in estimating voltage-gated conductance parameters were relatively minor (<10%). Thus in many cases isolation of coupled neurons may not be required if a small degree of measurement error of the voltage-gated currents or the synaptic potentials is acceptable. Modeling results show, however, that such errors may be as high as 20% if the gap-junction position is near the recording site or as high as 90% when measuring smaller voltage-gated ionic currents. Paradoxically, improved space clamp increases the errors arising from electrical coupling because voltage control across gap junctions is poor for even the highest realistic coupling conductances. Furthermore, the common procedure of leak subtraction can add an extra error to the conductance measurement, the sign of which depends on the maximal conductance.

Dynamic Action Potential Restitution Contributes to Mechanical Restitution in Right Ventricular Myocytes From Pulmonary Hypertensive Rats.

PubMed

Hardy, Matthew E L; Pervolaraki, Eleftheria; Bernus, Olivier; White, Ed

2018-01-01

We investigated the steepened dynamic action potential duration (APD) restitution of rats with pulmonary artery hypertension (PAH) and right ventricular (RV) failure and tested whether the observed APD restitution properties were responsible for negative mechanical restitution in these myocytes. PAH and RV failure were provoked in male Wistar rats by a single injection of monocrotaline (MCT) and compared with saline-injected animals (CON). Action potentials were recorded from isolated RV myocytes at stimulation frequencies between 1 and 9 Hz. Action potential waveforms recorded at 1 Hz were used as voltage clamp profiles (action potential clamp) at stimulation frequencies between 1 and 7 Hz to evoke rate-dependent currents. Voltage clamp profiles mimicking typical CON and MCT APD restitution were applied and cell shortening simultaneously monitored. Compared with CON myocytes, MCT myocytes were hypertrophied; had less polarized diastolic membrane potentials; had action potentials that were triggered by decreased positive current density and shortened by decreased negative current density; APD was longer and APD restitution steeper. APD90 restitution was unchanged by exposure to the late Na + -channel blocker (5 μM) ranolazine or the intracellular Ca 2+ buffer BAPTA. Under AP clamp, stimulation frequency-dependent inward currents were smaller in MCT myocytes and were abolished by BAPTA. In MCT myocytes, increasing stimulation frequency decreased contraction amplitude when depolarization duration was shortened, to mimic APD restitution, but not when depolarization duration was maintained. We present new evidence that the membrane potential of PAH myocytes is less stable than normal myocytes, being more easily perturbed by external currents. These observations can explain increased susceptibility to arrhythmias. We also present novel evidence that negative APD restitution is at least in part responsible for the negative mechanical restitution in PAH myocytes. Thus, our study links electrical restitution remodeling to a defining mechanical characteristic of heart failure, the reduced ability to respond to an increase in demand.

Can robots patch-clamp as well as humans? Characterization of a novel sodium channel mutation

PubMed Central

Estacion, M; Choi, J S; Eastman, E M; Lin, Z; Li, Y; Tyrrell, L; Yang, Y; Dib-Hajj, S D; Waxman, S G

2010-01-01

Ion channel missense mutations cause disorders of excitability by changing channel biophysical properties. As an increasing number of new naturally occurring mutations have been identified, and the number of other mutations produced by molecular approaches such as in situ mutagenesis has increased, the need for functional analysis by patch-clamp has become rate limiting. Here we compare a patch-clamp robot using planar-chip technology with human patch-clamp in a functional assessment of a previously undescribed Nav1.7 sodium channel mutation, S211P, which causes erythromelalgia. This robotic patch-clamp device can increase throughput (the number of cells analysed per day) by 3- to 10-fold. Both modes of analysis show that the mutation hyperpolarizes activation voltage dependence (−8 mV by manual profiling, −11 mV by robotic profiling), alters steady-state fast inactivation so that it requires an additional Boltzmann function for a second fraction of total current (∼20% manual, ∼40% robotic), and enhances slow inactivation (hyperpolarizing shift −15 mV by human, −13 mV robotic). Manual patch-clamping demonstrated slower deactivation and enhanced (∼2-fold) ramp response for the mutant channel while robotic recording did not, possibly due to increased temperature and reduced signal-to-noise ratio on the robotic platform. If robotic profiling is used to screen ion channel mutations, we recommend that each measurement or protocol be validated by initial comparison to manual recording. With this caveat, we suggest that, if results are interpreted cautiously, robotic patch-clamp can be used with supervision and subsequent confirmation from human physiologists to facilitate the initial profiling of a variety of electrophysiological parameters of ion channel mutations. PMID:20123784

Comparison of sarcolemmal calcium channel current in rabbit and rat ventricular myocytes.

PubMed Central

Yuan, W; Ginsburg, K S; Bers, D M

1996-01-01

1. Fundamental properties of Ca2+ channel currents in rat and rabbit ventricular myocytes were measured using whole cell voltage clamp. 2. In rat, as compared with rabbit myocytes, Ca2+ channel current (ICa) was half-activated at about 10 mV more negative potential, decayed slower, was half-inactivated (in steady state) at about 5 mV more positive potential, and recovered faster from inactivation. 3. These features result in a larger steady-state window current in rat, and also suggest that under comparable voltage clamp conditions, including action potential (AP) clamp, more Ca2+ influx would be expected in rat myocytes. 4. Ca2+ channel current carried by Na+ and Cs+ in the absence of divalent ions (Ins) also activated at more negative potential and decayed more slowly in rat. 5. The reversal potential for Ins was 6 mV more positive in rabbit, consistent with a larger permeability ratio (PNa/PCs) in rabbit than in rat. ICa also reversed at slightly more positive potentials in rabbit (such that PCa/PCs might also be higher). 6. Ca2+ influx was calculated by integration of ICa evoked by voltage clamp pulses (either square pulses or pulses based on recorded rabbit or rat APs). For a given clamp waveform, the Ca2+ influx was up to 25% greater in rat, as predicted from the fundamental properties of ICa and Ins. 7. However, the longer duration of the AP in rabbit myocytes compensated for the difference in influx, such that the integrated Ca2+ influx via ICa in response to the species-appropriate waveform was about twice as large as that seen in rat. PMID:8799895

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

Spontaneous activity of isolated dopaminergic periglomerular cells of the main olfactory bulb.

PubMed

Puopolo, Michelino; Bean, Bruce P; Raviola, Elio

2005-11-01

We examined the electrophysiological properties of a population of identified dopaminergic periglomerular cells of the main olfactory bulb using transgenic mice in which catecholaminergic neurons expressed human placental alkaline phosphatase (PLAP) on the outer surface of the plasma membrane. After acute dissociation, living dopaminergic periglomerular cells were identified by a fluorescently labeled monoclonal antibody to PLAP. In current-clamp mode, dopaminergic periglomerular cells spontaneously generated action potentials in a rhythmic fashion with an average frequency of 8 Hz. The hyperpolarization-activated cation current (Ih) did not seem important for pacemaking because blocking the current with ZD 7288 or Cs+ had little effect on spontaneous firing. To investigate what ionic currents do drive pacemaking, we performed action-potential-clamp experiments using records of pacemaking as voltage command in voltage-clamp experiments. We found that substantial TTX-sensitive Na+ current flows during the interspike depolarization. In addition, substantial Ca2+ current flowed during the interspike interval, and blocking Ca2+ current hyperpolarized the neurons and stopped spontaneous firing. These results show that dopaminergic periglomerular cells have intrinsic pacemaking activity, supporting the possibility that they can maintain a tonic release of dopamine to modulate the sensitivity of the olfactory system during odor detection. Calcium entry into these neurons provides electrical drive for pacemaking as well as triggering transmitter release.

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

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.

Robotic Automation of In Vivo Two-Photon Targeted Whole-Cell Patch-Clamp Electrophysiology.

PubMed

Annecchino, Luca A; Morris, Alexander R; Copeland, Caroline S; Agabi, Oshiorenoya E; Chadderton, Paul; Schultz, Simon R

2017-08-30

Whole-cell patch-clamp electrophysiological recording is a powerful technique for studying cellular function. While in vivo patch-clamp recording has recently benefited from automation, it is normally performed "blind," meaning that throughput for sampling some genetically or morphologically defined cell types is unacceptably low. One solution to this problem is to use two-photon microscopy to target fluorescently labeled neurons. Combining this with robotic automation is difficult, however, as micropipette penetration induces tissue deformation, moving target cells from their initial location. Here we describe a platform for automated two-photon targeted patch-clamp recording, which solves this problem by making use of a closed loop visual servo algorithm. Our system keeps the target cell in focus while iteratively adjusting the pipette approach trajectory to compensate for tissue motion. We demonstrate platform validation with patch-clamp recordings from a variety of cells in the mouse neocortex and cerebellum. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Effects of cevimeline on excitability of parasympathetic preganglionic neurons in the superior salivatory nucleus of rats.

PubMed

Mitoh, Yoshihiro; Ueda, Hirotaka; Ichikawa, Hiroyuki; Fujita, Masako; Kobashi, Motoi; Matsuo, Ryuji

2017-09-01

The superior salivatory nucleus (SSN) contains parasympathetic preganglionic neurons innervating the submandibular and sublingual salivary glands. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, is a sialogogue that possibly stimulates SSN neurons in addition to the salivary glands themselves because it can cross the blood-brain barrier (BBB). In the present study, we examined immunoreactivities for mAChR subtypes in SSN neurons retrogradely labeled with a fluorescent tracer in neonatal rats. Additionally, we examined the effects of cevimeline in labeled SSN neurons of brainstem slices using a whole-cell patch-clamp technique. Mainly M1 and M3 receptors were detected by immunohistochemical staining, with low-level detection of M4 and M5 receptors and absence of M2 receptors. Most (110 of 129) SSN neurons exhibited excitatory responses to application of cevimeline. In responding neurons, voltage-clamp recordings showed that 84% (101/120) of the neurons exhibited inward currents. In the neurons displaying inward currents, the effects of the mAChR antagonists were examined. A mixture of M1 and M3 receptor antagonists most effectively reduced the peak amplitude of inward currents, suggesting that the excitatory effects of cevimeline on SSN neurons were mainly mediated by M1 and M3 receptors. Current-clamp recordings showed that application of cevimeline induced membrane depolarization (9/9 neurons). These results suggest that most SSN neurons are excited by cevimeline via M1 and M3 muscarinic receptors. Copyright © 2017 Elsevier B.V. All rights reserved.

Sinusoidal voltage protocols for rapid characterisation of ion channel kinetics.

PubMed

Beattie, Kylie A; Hill, Adam P; Bardenet, Rémi; Cui, Yi; Vandenberg, Jamie I; Gavaghan, David J; de Boer, Teun P; Mirams, Gary R

2018-03-24

Ion current kinetics are commonly represented by current-voltage relationships, time constant-voltage relationships and subsequently mathematical models fitted to these. These experiments take substantial time, which means they are rarely performed in the same cell. Rather than traditional square-wave voltage clamps, we fitted a model to the current evoked by a novel sum-of-sinusoids voltage clamp that was only 8 s long. Short protocols that can be performed multiple times within a single cell will offer many new opportunities to measure how ion current kinetics are affected by changing conditions. The new model predicts the current under traditional square-wave protocols well, with better predictions of underlying currents than literature models. The current under a novel physiologically relevant series of action potential clamps is predicted extremely well. The short sinusoidal protocols allow a model to be fully fitted to individual cells, allowing us to examine cell-cell variability in current kinetics for the first time. Understanding the roles of ion currents is crucial to predict the action of pharmaceuticals and mutations in different scenarios, and thereby to guide clinical interventions in the heart, brain and other electrophysiological systems. Our ability to predict how ion currents contribute to cellular electrophysiology is in turn critically dependent on our characterisation of ion channel kinetics - the voltage-dependent rates of transition between open, closed and inactivated channel states. We present a new method for rapidly exploring and characterising ion channel kinetics, applying it to the hERG potassium channel as an example, with the aim of generating a quantitatively predictive representation of the ion current. We fitted a mathematical model to currents evoked by a novel 8 second sinusoidal voltage clamp in CHO cells overexpressing hERG1a. The model was then used to predict over 5 minutes of recordings in the same cell in response to further protocols: a series of traditional square step voltage clamps, and also a novel voltage clamp comprising a collection of physiologically relevant action potentials. We demonstrate that we can make predictive cell-specific models that outperform the use of averaged data from a number of different cells, and thereby examine which changes in gating are responsible for cell-cell variability in current kinetics. Our technique allows rapid collection of consistent and high quality data, from single cells, and produces more predictive mathematical ion channel models than traditional approaches. © 2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

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.

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.

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.

M-currents and other potassium currents in bullfrog sympathetic neurones

PubMed Central

Adams, P. R.; Brown, D. A.; Constanti, A.

1982-01-01

1. Bullfrog lumbar sympathetic neurones were voltage-clamped in vitro through twin micro-electrodes. Four different outward (K+) currents could be identified: (i) a large sustained voltage-sensitive delayed rectifier current (IK) activated at membrane potentials more positive than -25 mV; (ii) a calcium-dependent sustained outward current (IC) activated at similar positive potentials and peaking at +20 to +60 mV; (iii) a transient current (IA) activated at membrane potentials more positive than -60 mV after a hyperpolarizing pre-pulse, but which was rapidly and totally inactivated at all potentials within its activation range; and (iv) a new K+ current, the M-current (IM). 2. IM was detected as a non-inactivating current with a threshold at -60 mV. The underlying conductance GM showed a sigmoidal activation curve between -60 and -10 mV, with half-activation at -35 mV and a maximal value (ḠM) of 84±14 (S.E.M.) nS per neurone. The voltage sensitivity of GM could be expressed in terms of a simple Boltzmann distribution for a single multivalent gating particle. 3. IM activated and de-activated along an exponential time course with a time constant uniquely dependent upon voltage, maximizing at ≃ 150 ms at -35 mV at 22 °C. 4. Instantaneous current—voltage (I/V) curves were approximately linear in the presence of IM, suggesting that the M-channels do not show appreciable rectification. However, the time- and voltage-dependent opening of the M-channels induced considerable rectification in the steady-state I/V curves recorded under both voltage-clamp and current-clamp modes between -60 and -25 mV. Both time- and voltage-dependent rectification in the voltage responses to current injection over this range could be predicted from the kinetic properties of IM. 5. It is suggested that IM exerts a strong potential-clamping effect on the behaviour of these neurones at membrane potentials subthreshold to excitation. PMID:6294290

A nonlinear electrostatic potential change in the T-system of skeletal muscle detected under passive recording conditions using potentiometric dyes

PubMed Central

1990-01-01

Voltage-sensing dyes were used to examine the electrical behavior of the T-system under passive recording conditions similar to those commonly used to detect charge movement. These conditions are designed to eliminate all ionic currents and render the T-system potential linear with respect to the command potential applied at the surface membrane. However, we found an unexpected nonlinearity in the relationship between the dye signal from the T-system and the applied clamp potential. An additional voltage- and time-dependent optical signal appears over the same depolarizing range of potentials where change movement and mechanical activation occur. This nonlinearity is not associated with unblocked ionic currents and cannot be attributed to lack of voltage clamp control of the T-system, which appears to be good under these conditions. We propose that a local electrostatic potential change occurs in the T-system upon depolarization. An electrostatic potential would not be expected to extend beyond molecular distances of the membrane and therefore would be sensed by a charged dye in the membrane but not by the voltage clamp, which responds solely to the potential of the bulk solution. Results obtained with different dyes suggest that the location of the phenomena giving rise to the extra absorbance change is either intramembrane or at the inner surface of the T-system membrane. PMID:2299329

Presynaptic membrane potential affects transmitter release in an identified neuron in Aplysia by modulating the Ca2+ and K+ currents.

PubMed

Shapiro, E; Castellucci, V F; Kandel, E R

1980-01-01

We have examined the relationships between the modulation of transmitter release and of specific ionic currents by membrane potential in the cholinergic interneuron L10 of the abdominal ganglion of Aplysia californica. The presynaptic cell body was voltage-clamped under various pharmacological conditions and transmitter release from the terminals was assayed simultaneously by recording the synaptic potentials in the postsynaptic cell. When cell L10 was voltage-clamped from a holding potential of -60 mV in the presence of tetrodotoxin, graded transmitter release was evoked by depolarizing command pulses in the membrane voltage range (-35 mV to + 10 mV) in which the Ca(2+) current was also increasing. Depolarizing the holding potential of L10 results in increased transmitter output. Two ionic mechanisms contribute to this form of plasticity. First, depolarization inactivates some K(+) channels so that depolarizing command pulses recruit a smaller K(+) current. In unclamped cells the decreased K(+) conductance causes spike-broadening and increased influx of Ca(2+) during each spike. Second, small depolarizations around resting potential (-55 mV to -35 mV) activate a steady-state Ca(2+) current that also contributes to the modulation of transmitter release, because, even with most presynaptic K(+) currents blocked pharmacologically, depolarizing the holding potential still increases transmitter release. In contrast to the steady-state Ca(2+) current, the transient inward Ca(2+) current evoked by depolarizing clamp steps is relatively unchanged from various holding potentials.

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

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.

Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

PubMed Central

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

2013-01-01

Large-conductance voltage- and Ca2+-activated K+ (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca2+ imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca2+ levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca2+-dependent mechanism, thus increasing DSM contractility. PMID:24352333

The muscarinic inhibition of the potassium M-current modulates the action-potential discharge in the vestibular primary-afferent neurons of the rat.

PubMed

Pérez, C; Limón, A; Vega, R; Soto, E

2009-02-18

There is consensus that muscarinic and nicotinic receptors expressed in vestibular hair cells and afferent neurons are involved in the efferent modulation of the electrical activity of the afferent neurons. However the underlying mechanisms of postsynaptic control in neurons are not well understood. In our work we show that the activation of muscarinic receptors in the vestibular neurons modulates the potassium M-current modifying the activity of afferent neurons. Whole-cell patch-clamp recordings were made on vestibular-afferent neurons isolated from Wistar rats (postnatal days 7-10) and held in primary culture (18-24 h). The M-current was studied during its deactivation after depolarizing voltage-clamp pulses. In 68% of the cells studied, those of larger capacitance, the M-current antagonists linopirdine and XE-991 reduced the amplitude of the M-current by 54%+/-7% and 50%+/-3%. The muscarinic-receptor agonist oxotremorine-M also significantly reduced the M-current by 58%+/-12% in the cells. The action of oxotremorine-M was blocked by atropine, thus indicating its cholinergic nature. The erg-channel blocker E-4031 did not significantly modify the M-current amplitude. In current-clamp experiments, linopirdine, XE-991, and oxotremorine-M modified the discharge response to current pulses from single spike to multiple spiking, reducing the adaptation of the electrical discharge. Our results indicate that large soma-size cultured vestibular-afferent neurons (most probably calyx-bearing neurons) express the M-current and that the modulation of this current by activation of muscarinic-receptor reduces its spike-frequency adaptation.

An electrophysiological study on the effects of Pa-1G (a phospholipase A(2)) from the venom of king brown snake, Pseudechis australis, on neuromuscular function.

PubMed

Fatehi, M; Rowan, E G; Harvey, A L

2002-01-01

The effects of Pa-1G, a phospholipase A(2) (PLA(2)) from the venom of the Australian king brown snake (Pseudechis australis) were determined on the release of acetylcholine, muscle resting membrane potential and motor nerve terminal action potential at mouse neuromuscular junction. Intracellular recording from endplate regions of mouse triangularis sterni nerve-muscle preparations revealed that Pa-1G (800 nM) significantly reduced the amplitude of endplate potentials within 10 min exposure. The quantal content of endplate potentials was decreased to 58+/-6% of control after 30 min exposure to 800 nM Pa-1G. The toxin also caused a partial depolarisation of mouse muscle fibres within 60 min exposure. Extracellular recording of action potentials at motor nerve terminals showed that Pa-1G reduced the waveforms associated with both sodium and potassium conductances. To investigate whether this was a direct or indirect effect of the toxin on these ionic currents, whole cell patch clamp experiments were performed using human neuroblastoma (SK-N-SH) cells and B82 mouse fibroblasts stably transfected with rKv1.2. Patch clamp recording experiments confirmed that potassium currents sensitive to alpha-dendrotoxin recorded from B82 cells and sodium currents in SK-N-SH cells were not affected by the toxin. Since neither facilitation of acetylcholine release at mouse neuromuscular junction nor depression of potassium currents in B82 cells has been observed, the apparent blockade of potassium currents at mouse motor nerve endings induced by the toxin is unlikely to be due to a selective block of potassium channels.

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.

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.

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.

PKCɛ mediates substance P inhibition of GABAA receptors-mediated current in rat dorsal root ganglion.

PubMed

Li, Li; Zhao, Lei; Wang, Yang; Ma, Ke-tao; Shi, Wen-yan; Wang, Ying-zi; Si, Jun-qiang

2015-02-01

The mechanism underlying the modulatory effect of substance P (SP) on GABA-activated response in rat dorsal root ganglion (DRG) neurons was investigated. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique was used to record GABA-activated current and sharp electrode intracellular recording technique was used to record GABA-induced membrane depolarization. Application of GABA (1-1000 μmol/L) induced an inward current in a concentration-dependent manner in 114 out of 127 DRG neurons (89.8 %) examined with whole-cell patch-clamp recordings. Bath application of GABA (1-1000 μmol/L) evoked a depolarizing response in 236 out of 257 (91.8%) DRG neurons examined with intracellular recordings. Application of SP (0.001-1 μmol/L) suppressed the GABA-activated inward current and membrane depolarization. The inhibitory effects were concentration-dependent and could be blocked by the selective neurokinin 1 (NK1) receptors antagonist spantide but not by L659187 and SR142801 (1 μmol/L, n=7), selective antagonists of NK2 and NK3. The inhibitory effect of SP was significantly reduced by the calcium chelator BAPTA-AM, phospholipase C (PLC) inhibitor U73122, and PKC inhibitor chelerythrine, respectively. The PKA inhibitor H-89 did not affect the SP effect. Remarkably, the inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKCε inhibitor epilon-V1-2 but not by safingol and LY333531, selective inhibitors of PKCα and PKCβ. Our results suggest that NK1 receptor mediates SP-induced inhibition of GABA-activated current and membrane depolarization by activating intracellular PLC-Ca²⁺-PKCε cascade. SP might regulate the excitability of peripheral nociceptors through inhibition of the "pre-synaptic inhibition" evoked by GABA, which may explain its role in pain and neurogenic inflammation.

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

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

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

Preferential inhibition of Ih in rat trigeminal ganglion neurons by an organic blocker.

PubMed

Janigro, D; Martenson, M E; Baumann, T K

1997-11-15

The potency and specificity of a novel organic Ih current blocker DK-AH 268 (DK, Boehringer) was studied in cultured rat trigeminal ganglion neurons using whole-cell patch-clamp recording techniques. In neurons current-clamped at the resting potential, the application of 10 microM DK caused a slight hyperpolarization of the membrane potential and a small increase in the threshold for action potential discharge without any major change in the shape of the action potential. In voltage-clamped neurons, DK caused a reduction of a hyperpolarization-activated current. Current subtraction protocols revealed that the time-dependent, hyperpolarization-activated currents blocked by 10 microM DK or external Cs+ (3 mM) had virtually identical activation properties, suggesting that DK and Cs+ caused blockade of the same current, namely Ih. The block of Ih by DK was dose-dependent. At the intermediate and higher concentrations of DK (10 and 100 microM) a decrease in specificity was observed so that time-independent, inwardly rectifying and noninactivating, voltage-gated outward potassium currents were also reduced by DK but to a much lesser extent than the time-dependent, hyperpolarization-activated currents. Blockade of the time-dependent, hyperpolarization-activated currents by DK appeared to be use-dependent since it required hyperpolarization for the effect to take place. Relief of DK block was also aided by membrane hyperpolarization. Since both the time-dependent current blocked by DK and the Cs+-sensitive time-dependent current behaved as Ih, we conclude that 10 microM DK can preferentially reduce Ih without a major effect on other potassium currents. Thus, DK may be a useful agent in the investigation of the function of Ih in neurons.

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.

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

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.

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

Isoflurane modulates neuronal excitability of the nucleus reticularis thalami in vitro.

PubMed

Joksovic, Pavle M; Todorovic, Slobodan M

2010-06-01

The thalamus has a key function in processing sensory information, sleep, and cognition. We examined the effects of a common volatile anesthetic, isoflurane, on modulation of neuronal excitability in reticular thalamic nucleus (nRT) in intact brain slices from immature rats. In current-clamp recordings, isoflurane (300-600 micromol/L) consistently depolarized membrane potential, decreased input resistance, and inhibited both rebound burst firing and tonic spike firing modes of nRT neurons. The isoflurane-induced depolarization persisted not only in the presence of tetrodotoxin, but after replacement of Ca(2+) with Ba(2+) ions in external solution; it was abolished by partial replacement of extracellular Na(+) ions with N-methyl-D-glucamine. In voltage-clamp recordings, we found that isoflurane slowed recovery from inactivation of T-type Ca(2+) current. Thus, at clinically relevant concentrations, isoflurane inhibits neuronal excitability of nRT neurons in developing brain via multiple ion channels. Inhibition of the neuronal excitability of thalamic cells may contribute to impairment of sensory information transfer in the thalamocortical network by general anesthetics. The findings may be important for understanding cellular mechanisms of anesthesia, such as loss of consciousness and potentially damaging consequences of general anesthetics on developing mammalian brains.

Isolation and culture of adult mouse vestibular nucleus neurons

PubMed

Him, Aydın; Altuntaş, Serap; Öztürk, Gürkan; Erdoğan, Ender; Cengiz, Nureddin

2017-12-19

Background/aim: Isolated cell cultures are widely used to study neuronal properties due to their advantages. Although embryonic animals are preferred for culturing, their morphological or electrophysiological properties may not reflect adult neurons, which may be important in neurodegenerative diseases. This paper aims to develop a method for preparing isolated cell cultures of medial vestibular nucleus (MVN) from adult mice and describe its morphological and electrophysiological properties.Materials and methods: Vestibular nucleus neurons were mechanically and enzymatically isolated and cultured using a defined medium with known growth factors. Cell survival was measured with propidium iodide, and electrophysiological properties were investigated with current-clamp recording.Results: Vestibular neurons grew neurites in cultures, gaining adult-like morphological properties, and stayed viable for 3 days in culture. Adding bovine calf serum, nerve growth factor, or insulin-like growth factor into the culture medium enhanced neuronal viability. Current-clamp recording of the cultured neurons revealed tonic and phasic-type neurons with similar input resistance, resting membrane potential, action potential amplitude, and duration. Conclusion: Vestibular neurons from adult mice can be cultured, and regenerate axons in a medium containing appropriate growth factors. Culturing adult vestibular neurons provides a new method to study age-related pathologies of the vestibular system.

Sodium and potassium conductance changes during a membrane action potential.

PubMed

Bezanilla, F; Rojas, E; Taylor, R E

1970-12-01

1. A method for turning a membrane potential control system on and off in less than 10 musec is described. This method was used to record membrane currents in perfused giant axons from Dosidicus gigas and Loligo forbesi after turning on the voltage clamp system at various times during the course of a membrane action potential.2. The membrane current measured just after the capacity charging transient was found to have an almost linear relation to the controlled membrane potential.3. The total membrane conductance taken from these current-voltage curves was found to have a time course during the action potential similar to that found by Cole & Curtis (1939).4. The instantaneous current voltage curves were linear enough to make it possible to obtain a good estimate of the individual sodium and potassium channel conductances, either algebraically or by clamping to the sodium, or potassium, reversal potentials. Good general agreement was obtained with the predictions of the Hodgkin-Huxley equations.5. We consider these results to constitute the first direct experimental demonstration of the conductance changes to sodium and potassium during the course of an action potential.

Payload Launch Lock Mechanism

NASA Technical Reports Server (NTRS)

Young, Ken (Inventor); Hindle, Timothy (Inventor)

2014-01-01

A payload launch lock mechanism includes a base, a preload clamp, a fastener, and a shape memory alloy (SMA) actuator. The preload clamp is configured to releasibly restrain a payload. The fastener extends, along an axis, through the preload clamp and into the base, and supplies a force to the preload clamp sufficient to restrain the payload. The SMA actuator is disposed between the base and the clamp. The SMA actuator is adapted to receive electrical current and is configured, upon receipt of the electrical current, to supply a force that causes the fastener to elongate without fracturing. The preload clamp, in response to the fastener elongation, either rotates or pivots to thereby release the payload.

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

Na+ current in presynaptic terminals of the crayfish opener cannot initiate action potentials.

PubMed

Lin, Jen-Wei

2016-01-01

Action potential (AP) propagation in presynaptic axons of the crayfish opener neuromuscular junction (NMJ) was investigated by simultaneously recording from a terminal varicosity and a proximal branch. Although orthodromically conducting APs could be recorded in terminals with amplitudes up to 70 mV, depolarizing steps in terminals to -20 mV or higher failed to fire APs. Patch-clamp recordings did detect Na(+) current (INa) in most terminals. The INa exhibited a high threshold and fast activation rate. Local perfusion of Na(+)-free saline showed that terminal INa contributed to AP waveform by slightly accelerating the rising phase and increasing the peak amplitude. These findings suggest that terminal INa functions to "touch up" but not to generate APs. Copyright © 2016 the American Physiological Society.

Expression of a functional hyperpolarization-activated current (Ih) in the mouse nucleus reticularis thalami.

PubMed

Rateau, Y; Ropert, N

2006-05-01

The GABAergic neurons of the nucleus reticularis thalami (nRT) express the type 2 hyperpolarization-activated cAMP-sensitive (HCN2) subunit mRNA, but surprisingly, they were reported to lack the hyperpolarization-activated (Ih) current carried by this subunit. Using the voltage-clamp recordings in the thalamocortical slice preparation of the newborn and juvenile mice (P6-P23), we demonstrate that, in the presence of 1 mM barium (Ba2+), the nRT neurons express a slow hyperpolarization-activated inward current, suggesting that the Ih is present but masked in control conditions by K+ leak currents. We investigate the identity of the hyperpolarization-activated current in the nRT by studying its physiological and pharmacological profile in presence of Ba2+. We show that it has voltage- and time-dependent properties typical of the Ih, that it is blocked by cesium and ZD7288, two blockers of the Ih, and that it is carried both by the K+ and Na+ ions. We could also alter the gating characteristics of the hyperpolarization-activated current in the nRT by adding a nonhydrolysable analogue of cAMP to the pipette solution. Finally, using the current-clamp recording, we showed that blocking the hyperpolarization-activated current induced an hyperpolarization correlated with an increase of the R(in) of the nRT neurons. In conclusion, our results demonstrate that the nRT neurons express the Ih with slow kinetics similar to those described for the homomeric HCN2 channels, and we show that the Ih of the nRT contributes to the excitability of the nRT neurons in normal conditions.

Physiological roles of Kv2 channels in entorhinal cortex layer II stellate cells revealed by Guangxitoxin‐1E

PubMed Central

Hönigsperger, Christoph; Nigro, Maximiliano J.

2016-01-01

Key points Kv2 channels underlie delayed‐rectifier potassium currents in various neurons, although their physiological roles often remain elusive. Almost nothing is known about Kv2 channel functions in medial entorhinal cortex (mEC) neurons, which are involved in representing space, memory formation, epilepsy and dementia.Stellate cells in layer II of the mEC project to the hippocampus and are considered to be space‐representing grid cells. We used the new Kv2 blocker Guangxitoxin‐1E (GTx) to study Kv2 functions in these neurons.Voltage clamp recordings from mEC stellate cells in rat brain slices showed that GTx inhibited delayed‐rectifier K+ current but not transient A‐type current.In current clamp, GTx had multiple effects: (i) increasing excitability and bursting at moderate spike rates but reducing firing at high rates; (ii) enhancing after‐depolarizations; (iii) reducing the fast and medium after‐hyperpolarizations; (iv) broadening action potentials; and (v) reducing spike clustering.GTx is a useful tool for studying Kv2 channels and their functions in neurons. Abstract The medial entorhinal cortex (mEC) is strongly involved in spatial navigation, memory, dementia and epilepsy. Although potassium channels shape neuronal activity, their roles in mEC are largely unknown. We used the new Kv2 blocker Guangxitoxin‐1E (GTx; 10–100 nm) in rat brain slices to investigate Kv2 channel functions in mEC layer II stellate cells (SCs). These neurons project to the hippocampus and are considered to be grid cells representing space. Voltage clamp recordings from SCs nucleated patches showed that GTx inhibited a delayed rectifier K+ current activating beyond –30 mV but not transient A‐type current. In current clamp, GTx (i) had almost no effect on the first action potential but markedly slowed repolarization of late spikes during repetitive firing; (ii) enhanced the after‐depolarization (ADP); (iii) reduced fast and medium after‐hyperpolarizations (AHPs); (iv) strongly enhanced burst firing and increased excitability at moderate spike rates but reduced spiking at high rates; and (v) reduced spike clustering and rebound potentials. The changes in bursting and excitability were related to the altered ADPs and AHPs. Kv2 channels strongly shape the activity of mEC SCs by affecting spike repolarization, after‐potentials, excitability and spike patterns. GTx is a useful tool and may serve to further clarify Kv2 channel functions in neurons. We conclude that Kv2 channels in mEC SCs are important determinants of intrinsic properties that allow these neurons to produce spatial representation. The results of the present study may also be important for the accurate modelling of grid cells. PMID:27562026

The effects of some inhalation anaesthetics on the sodium current of the squid giant axon.

PubMed Central

Haydon, D A; Urban, B W

1983-01-01

The effects of diethyl ether, methoxyflurane, halothane, dichloromethane and chloroform on the ionic currents and electrical capacity of the squid giant axon have been examined. The peak inward current in voltage-clamped axons was reduced reversibly by each substance. Sodium currents under voltage clamp were recorded in intracellularly perfused axons before, during, and sometimes after exposure to the test substances, and the records were fitted with equations similar to those proposed by Hodgkin & Huxley (1952). Shifts in the dependence of the steady-state activation and inactivation parameters (m infinity and h infinity) on membrane potential, reductions in the peak heights of the activation and inactivation time constants (tau m and tau h) and decreases in the maximum Na conductance (gNa) have been tabulated. For each of the anaesthetics the steady-state inactivation curve was shifted in the hyperpolarizing direction though less markedly than for the hydrocarbons. The steady-state activation curve was in each instance shifted in the depolarizing direction, as for the alcohols and other surface active substances. In common with both the hydrocarbons and the surface active substances the peak time constants were invariably reduced. The membrane capacity at 100 kHz was affected significantly only by methoxyflurane, where decreases of ca. 9% were observed for 3 mM solutions. The extent to which the results can be accounted for in terms of the perturbation of membrane lipid has been discussed. PMID:6312031

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.

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

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.

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.

Nitric oxide augments voltage-activated calcium currents of crustacea (Idotea baltica) skeletal muscle.

PubMed

Erxleben, C; Hermann, A

2001-03-16

Invertebrate skeletal muscle contraction is regulated by calcium influx through voltage-dependent calcium channels in the sarcolemmal membrane. In present study we investigated the effects of nitric oxide (NO) donors on calcium currents of single skeletal muscle fibres from the marine isopod, Idotea baltica, using two-electrode voltage clamp recording techniques. The NO donors, S-nitrosocysteine, S-nitroso-N-acetyl-penicillamine or hydroxylamine reversibly increased calcium inward currents in a time dependent manner. The increase of the current was prevented by methylene blue. Our experiments suggest that NO increases calcium inward currents. NO, by acting on calcium ion channels in the sarcolemmal membrane, therefore, may directly be involved in the modulation of muscle contraction.

[Protective effect of Uncaria rhynchophylla total alkaloids pretreatment on hippocampal neurons after acute hypoxia].

PubMed

Liu, Wei; Zhang, Zhao-qin; Zhao, Xiao-min; Gao, Yun-sheng

2006-05-01

To investigate the effect of Uncaria rhynchophylla total alkaloids (RTA) pretreatment on the voltage-gated sodium currents of the rat hippocampal neurons after acute hypoxia. Primary cultured hippocampal neurons were divided into RTA pre-treated and non-pretreated groups. Patch clamp whole-cell recording was used to compare the voltage-gated sodium current amplitude and threshold with those before hypoxia. After acute hypoxia, sodium current amplitude was significantly decreased and its threshold was upside. RTA pretreatment could inhibit the reduction of sodium current amplitude. RTA pretreatment alleviates the acute hypoxia-induced change of sodium currents, which may be one of the mechanisms for protective effect of RTA on cells.

Quantitative analysis of the Ca2+ -dependent regulation of delayed rectifier K+ current IKs in rabbit ventricular myocytes.

PubMed

Bartos, Daniel C; Morotti, Stefano; Ginsburg, Kenneth S; Grandi, Eleonora; Bers, Donald M

2017-04-01

[Ca 2+ ] i enhanced rabbit ventricular slowly activating delayed rectifier K + current (I Ks ) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K + current (I Kr ) amplitude and voltage dependence were unaffected by high [Ca 2+ ] i . When measuring or simulating I Ks during an action potential, I Ks was not different during a physiological Ca 2+ transient or when [Ca 2+ ] i was buffered to 500 nm. The slowly activating delayed rectifier K + current (I Ks ) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca 2+ ([Ca 2+ ] i ) and β-adrenergic receptor (β-AR) stimulation modulate I Ks amplitude and kinetics, but details of these important I Ks regulators and their interaction are limited. We assessed the [Ca 2+ ] i dependence of I Ks in steady-state conditions and with dynamically changing membrane potential and [Ca 2+ ] i during an AP. I Ks was recorded from freshly isolated rabbit ventricular myocytes using whole-cell patch clamp. With intracellular pipette solutions that controlled free [Ca 2+ ] i , we found that raising [Ca 2+ ] i from 100 to 600 nm produced similar increases in I Ks as did β-AR activation, and the effects appeared additive. Both β-AR activation and high [Ca 2+ ] i increased maximally activated tail I Ks , negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well-established mathematical model of the rabbit myocyte. In both AP-clamp experiments and simulations, I Ks recorded during a normal physiological Ca 2+ transient was similar to I Ks measured with [Ca 2+ ] i clamped at 500-600 nm. Thus, our study provides novel quantitative data as to how physiological [Ca 2+ ] i regulates I Ks amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca 2+ ] i , in the submembrane or junctional cleft space, is not required to maximize [Ca 2+ ] i -dependent I Ks activation during normal Ca 2+ transients. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Quantitative analysis of the Ca2+‐dependent regulation of delayed rectifier K+ current I Ks in rabbit ventricular myocytes

PubMed Central

Bartos, Daniel C.; Morotti, Stefano; Ginsburg, Kenneth S.; Grandi, Eleonora

2017-01-01

Key points [Ca2+]i enhanced rabbit ventricular slowly activating delayed rectifier K+ current (I Ks) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol.Rabbit ventricular rapidly activating delayed rectifier K+ current (I Kr) amplitude and voltage dependence were unaffected by high [Ca2+]i.When measuring or simulating I Ks during an action potential, I Ks was not different during a physiological Ca2+ transient or when [Ca2+]i was buffered to 500 nm. Abstract The slowly activating delayed rectifier K+ current (I Ks) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca2+ ([Ca2+]i) and β‐adrenergic receptor (β‐AR) stimulation modulate I Ks amplitude and kinetics, but details of these important I Ks regulators and their interaction are limited. We assessed the [Ca2+]i dependence of I Ks in steady‐state conditions and with dynamically changing membrane potential and [Ca2+]i during an AP. I Ks was recorded from freshly isolated rabbit ventricular myocytes using whole‐cell patch clamp. With intracellular pipette solutions that controlled free [Ca2+]i, we found that raising [Ca2+]i from 100 to 600 nm produced similar increases in I Ks as did β‐AR activation, and the effects appeared additive. Both β‐AR activation and high [Ca2+]i increased maximally activated tail I Ks, negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well‐established mathematical model of the rabbit myocyte. In both AP‐clamp experiments and simulations, I Ks recorded during a normal physiological Ca2+ transient was similar to I Ks measured with [Ca2+]i clamped at 500–600 nm. Thus, our study provides novel quantitative data as to how physiological [Ca2+]i regulates I Ks amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca2+]i, in the submembrane or junctional cleft space, is not required to maximize [Ca2+]i‐dependent I Ks activation during normal Ca2+ transients. PMID:28008618

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

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.

An accurate online calibration system based on combined clamp-shape coil for high voltage electronic current transformers.

PubMed

Li, Zhen-hua; Li, Hong-bin; Zhang, Zhi

2013-07-01

Electronic transformers are widely used in power systems because of their wide bandwidth and good transient performance. However, as an emerging technology, the failure rate of electronic transformers is higher than that of traditional transformers. As a result, the calibration period needs to be shortened. Traditional calibration methods require the power of transmission line be cut off, which results in complicated operation and power off loss. This paper proposes an online calibration system which can calibrate electronic current transformers without power off. In this work, the high accuracy standard current transformer and online operation method are the key techniques. Based on the clamp-shape iron-core coil and clamp-shape air-core coil, a combined clamp-shape coil is designed as the standard current transformer. By analyzing the output characteristics of the two coils, the combined clamp-shape coil can achieve verification of the accuracy. So the accuracy of the online calibration system can be guaranteed. Moreover, by employing the earth potential working method and using two insulating rods to connect the combined clamp-shape coil to the high voltage bus, the operation becomes simple and safe. Tests in China National Center for High Voltage Measurement and field experiments show that the proposed system has a high accuracy of up to 0.05 class.

Redox artifacts in electrophysiological recordings

PubMed Central

Berman, Jonathan M.

2013-01-01

Electrophysiological techniques make use of Ag/AgCl electrodes that are in direct contact with cells or bath. In the bath, electrodes are exposed to numerous experimental conditions and chemical reagents that can modify electrode voltage. We examined voltage offsets created in Ag/AgCl electrodes by exposure to redox reagents used in electrophysiological studies. Voltage offsets were measured in reference to an electrode separated from the solution by an agar bridge. The reducing reagents Tris-2-carboxyethly-phosphine, dithiothreitol (DTT), and glutathione, as well as the oxidizing agent H2O2 used at experimentally relevant concentrations reacted with Ag in the electrodes to produce voltage offsets. Chloride ions and strong acids and bases produced offsets at millimolar concentrations. Electrolytic depletion of the AgCl layer, to replicate voltage clamp and sustained use, resulted in increased sensitivity to flow and DTT. Offsets were sensitive to electrode silver purity and to the amount and method of chloride deposition. For example, exposure to 10 μM DTT produced a voltage offset between 10 and 284 mV depending on the chloride deposition method. Currents generated by these offsets are significant and dependent on membrane conductance and by extension the expression of ion channels and may therefore appear to be biological in origin. These data demonstrate a new source of artifacts in electrophysiological recordings that can affect measurements obtained from a variety of experimental techniques from patch clamp to two-electrode voltage clamp. PMID:23344161

Depolarized inactivation overcomes impaired activation to produce DRG neuron hyperexcitability in a Nav1.7 mutation in a patient with distal limb pain.

PubMed

Huang, Jianying; Yang, Yang; Dib-Hajj, Sulayman D; van Es, Michael; Zhao, Peng; Salomon, Jody; Drenth, Joost P H; Waxman, Stephen G

2014-09-10

Sodium channel Nav1.7, encoded by SCN9A, is expressed in DRG neurons and regulates their excitability. Genetic and functional studies have established a critical contribution of Nav1.7 to human pain disorders. We have now characterized a novel Nav1.7 mutation (R1279P) from a female human subject with distal limb pain, in which depolarized fast inactivation overrides impaired activation to produce hyperexcitability and spontaneous firing in DRG neurons. Whole-cell voltage-clamp recordings in human embryonic kidney (HEK) 293 cells demonstrated that R1279P significantly depolarizes steady-state fast-, slow-, and closed-state inactivation. It accelerates deactivation, decelerates inactivation, and facilitates repriming. The mutation increases ramp currents in response to slow depolarizations. Our voltage-clamp analysis showed that R1279P depolarizes channel activation, a change that was supported by our multistate structural modeling. Because this mutation confers both gain-of-function and loss-of-function attributes on the Nav1.7 channel, we tested the impact of R1279P expression on DRG neuron excitability. Current-clamp studies reveal that R1279P depolarizes resting membrane potential, decreases current threshold, and increases firing frequency of evoked action potentials within small DRG neurons. The populations of spontaneously firing and repetitively firing neurons were increased by expressing R1279P. These observations indicate that the dominant proexcitatory gating changes associated with this mutation, including depolarized steady-state fast-, slow-, and closed-state inactivation, faster repriming, and larger ramp currents, override the depolarizing shift of activation, to produce hyperexcitability and spontaneous firing of nociceptive neurons that underlie pain. Copyright © 2014 the authors 0270-6474/14/3412328-13$15.00/0.

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.

Effects of premature stimulation on HERG K+ channels

PubMed Central

Lu, Yu; Mahaut-Smith, Martyn P; Varghese, Anthony; Huang, Christopher L-H; Kemp, Paul R; Vandenberg, Jamie I

2001-01-01

The unusual kinetics of human ether-à-go-go-related gene (HERG) K+ channels are consistent with a role in the suppression of arrhythmias initiated by premature beats. Action potential clamp protocols were used to investigate the effect of premature stimulation on HERG K+ channels, transfected in Chinese hamster ovary cells, at 37 °C. HERG K+ channel currents peaked during the terminal repolarization phase of normally paced action potential waveforms. However, the magnitude of the current and the time point at which conductance was maximal depended on the type of action potential waveform used (epicardial, endocardial, Purkinje fibre or atrial). HERG K+ channel currents recorded during premature action potentials consisted of an early transient outward current followed by a sustained outward current. The magnitude of the transient current component showed a biphasic dependence on the coupling interval between the normally paced and premature action potentials and was maximal at a coupling interval equivalent to 90% repolarization (APD90) for ventricular action potentials. The largest transient current response occurred at shorter coupling intervals for Purkinje fibre (APD90– 20 ms) and atrial (APD90– 30 ms) action potentials. The magnitude of the sustained current response following premature stimulation was similar to that recorded during the first action potential for ventricular action potential waveforms. However, for Purkinje and atrial action potentials the sustained current response was significantly larger during the premature action potential than during the normally paced action potential. A Markov model that included three closed states, one open and one inactivated state with transitions permitted between the pre-open closed state and the inactivated state, successfully reproduced our results for the effects of premature stimuli, both during square pulse and action potential clamp waveforms. These properties of HERG K+ channels may help to suppress arrhythmias initiated by early afterdepolarizations and premature beats in the ventricles, Purkinje fibres or atria. PMID:11744759

Sodium and potassium conductance changes during a membrane action potential

PubMed Central

Bezanilla, Francisco; Rojas, Eduardo; Taylor, Robert E.

1970-01-01

1. A method for turning a membrane potential control system on and off in less than 10 μsec is described. This method was used to record membrane currents in perfused giant axons from Dosidicus gigas and Loligo forbesi after turning on the voltage clamp system at various times during the course of a membrane action potential. 2. The membrane current measured just after the capacity charging transient was found to have an almost linear relation to the controlled membrane potential. 3. The total membrane conductance taken from these current—voltage curves was found to have a time course during the action potential similar to that found by Cole & Curtis (1939). 4. The instantaneous current voltage curves were linear enough to make it possible to obtain a good estimate of the individual sodium and potassium channel conductances, either algebraically or by clamping to the sodium, or potassium, reversal potentials. Good general agreement was obtained with the predictions of the Hodgkin—Huxley equations. 5. We consider these results to constitute the first direct experimental demonstration of the conductance changes to sodium and potassium during the course of an action potential. PMID:5505231

Carbachol regulates pacemaker activities in cultured interstitial cells of Cajal from the mouse small intestine.

PubMed

So, Keum Young; Kim, Sang Hun; Sohn, Hong Moon; Choi, Soo Jin; Parajuli, Shankar Prasad; Choi, Seok; Yeum, Cheol Ho; Yoon, Pyung Jin; Jun, Jae Yeoul

2009-05-31

We studied the effect of carbachol on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine by muscarinic stimulation using a whole cell patch clamp technique and Ca2+-imaging. ICC generated periodic pacemaker potentials in the current-clamp mode and generated spontaneous inward pacemaker currents at a holding potential of-70 mV. Exposure to carbachol depolarized the membrane and produced tonic inward pacemaker currents with a decrease in the frequency and amplitude of the pacemaker currents. The effects of carbachol were blocked by 1-dimethyl-4-diphenylacetoxypiperidinium, a muscarinic M(3) receptor antagonist, but not by methotramine, a muscarinic M(2) receptor antagonist. Intracellular GDP-beta-S suppressed the carbachol-induced effects. Carbachol-induced effects were blocked by external Na+-free solution and by flufenamic acid, a non-selective cation channel blocker, and in the presence of thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum. However, carbachol still produced tonic inward pacemaker currents with the removal of external Ca2+. In recording of intracellular Ca2+ concentrations using fluo 3-AM dye, carbachol increased intracellular Ca2+ concentrations with increasing of Ca2+ oscillations. These results suggest that carbachol modulates the pacemaker activity of ICC through the activation of non-selective cation channels via muscarinic M(3) receptors by a G-protein dependent intracellular Ca2+ release mechanism.

An accurate online calibration system based on combined clamp-shape coil for high voltage electronic current transformers

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

Li, Zhen-hua; Li, Hong-bin; Zhang, Zhi

Electronic transformers are widely used in power systems because of their wide bandwidth and good transient performance. However, as an emerging technology, the failure rate of electronic transformers is higher than that of traditional transformers. As a result, the calibration period needs to be shortened. Traditional calibration methods require the power of transmission line be cut off, which results in complicated operation and power off loss. This paper proposes an online calibration system which can calibrate electronic current transformers without power off. In this work, the high accuracy standard current transformer and online operation method are the key techniques. Basedmore » on the clamp-shape iron-core coil and clamp-shape air-core coil, a combined clamp-shape coil is designed as the standard current transformer. By analyzing the output characteristics of the two coils, the combined clamp-shape coil can achieve verification of the accuracy. So the accuracy of the online calibration system can be guaranteed. Moreover, by employing the earth potential working method and using two insulating rods to connect the combined clamp-shape coil to the high voltage bus, the operation becomes simple and safe. Tests in China National Center for High Voltage Measurement and field experiments show that the proposed system has a high accuracy of up to 0.05 class.« less

Elevated Neuronal Excitability Due to Modulation of the Voltage-Gated Sodium Channel Nav1.6 by Aβ1−42

PubMed Central

Wang, Xi; Zhang, Xiao-Gang; Zhou, Ting-Ting; Li, Na; Jang, Chun-Yan; Xiao, Zhi-Cheng; Ma, Quan-Hong; Li, Shao

2016-01-01

Aberrant increases in neuronal network excitability may contribute to the cognitive deficits in Alzheimer's disease (AD). However, the mechanisms underlying hyperexcitability are not fully understood. Such overexcitation of neuronal networks has been detected in the brains of APP/PS1 mice. In the present study, using current-clamp recording techniques, we observed that 12 days in vitro (DIV) primary cultured pyramidal neurons from P0 APP/PS1 mice exhibited a more prominent action potential burst and a lower threshold than WT littermates. Moreover, after treatment with Aβ1−42 peptide, 12 DIV primary cultured neurons showed similar changes, to a greater degree than in controls. Voltage-clamp recordings revealed that the voltage-dependent sodium current density of neurons incubated with Aβ1−42 was significantly increased, without change in the voltage-dependent sodium channel kinetic characteristics. Immunohistochemistry and western blot results showed that, after treatment with Aβ1−42, expressions of Nav and Nav1.6 subtype increased in cultured neurons or APP/PS1 brains compared to control groups. The intrinsic neuronal hyperexcitability of APP/PS1 mice might thus be due to an increased expression of voltage-dependent sodium channels induced by Aβ1−42. These results may illuminate the mechanism of aberrant neuronal networks in AD. PMID:27013956

Octopamine increases the excitability of neurons in the snail feeding system by modulation of inward sodium current but not outward potassium currents.

PubMed

Vehovszky, Agnes; Szabó, Henriette; Elliott, Christopher J H

2005-12-06

Although octopamine has long been known to have major roles as both transmitter and modulator in arthropods, it has only recently been shown to be functionally important in molluscs, playing a role as a neurotransmitter in the feeding network of the snail Lymnaea stagnalis. The synaptic potentials cannot explain all the effects of octopamine-containing neurons on the feeding network, and here we test the hypothesis that octopamine is also a neuromodulator. The excitability of the B1 and B4 motoneurons in the buccal ganglia to depolarising current clamp pulses is significantly (P < < 0.05) increased by (10 microM) octopamine, whereas the B2 motoneuron becomes significantly less excitable. The ionic currents evoked by voltage steps were recorded using 2-electrode voltage clamp. The outward current of B1, B2 and B4 motoneurons had two components, a transient IA current and a sustained IK delayed-rectifier current, but neither was modulated by octopamine in any of these three buccal neurons. The fast inward current was eliminated in sodium-free saline and so is likely to be carried by sodium ions. 10 microM octopamine enhanced this current by 33 and 45% in the B1 and B4 motoneurons respectively (P < < 0.05), but a small reduction was seen in the B2 neuron. A Hodgkin-Huxley style simulation of the B1 motoneuron confirms that a 33% increase in the fast inward current by octopamine increases the excitability markedly. We conclude that octopamine is also a neuromodulator in snails, changing the excitability of the buccal neurons. This is supported by the close relationship from the voltage clamp data, through the quantitative simulation, to the action potential threshold, changing the properties of neurons in a rhythmic network. The increase in inward sodium current provides an explanation for the polycyclic modulation of the feeding system by the octopamine-containing interneurons, making feeding easier to initiate and making the feeding bursts more intense.

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.

Naturalistic stimulation changes the dynamic response of action potential encoding in a mechanoreceptor

PubMed Central

Pfeiffer, Keram; French, Andrew S.

2015-01-01

Naturalistic signals were created from vibrations made by locusts walking on a Sansevieria plant. Both naturalistic and Gaussian noise signals were used to mechanically stimulate VS-3 slit-sense mechanoreceptor neurons of the spider, Cupiennius salei, with stimulus amplitudes adjusted to give similar firing rates for either stimulus. Intracellular microelectrodes recorded action potentials, receptor potential, and receptor current, using current clamp and voltage clamp. Frequency response analysis showed that naturalistic stimulation contained relatively more power at low frequencies, and caused increased neuronal sensitivity to higher frequencies. In contrast, varying the amplitude of Gaussian stimulation did not change neuronal dynamics. Naturalistic stimulation contained less entropy than Gaussian, but signal entropy was higher than stimulus in the resultant receptor current, indicating addition of uncorrelated noise during transduction. The presence of added noise was supported by measuring linear information capacity in the receptor current. Total entropy and information capacity in action potentials produced by either stimulus were much lower than in earlier stages, and limited to the maximum entropy of binary signals. We conclude that the dynamics of action potential encoding in VS-3 neurons are sensitive to the form of stimulation, but entropy and information capacity of action potentials are limited by firing rate. PMID:26578975

Voltage-Clamp Studies on Uterine Smooth Muscle

PubMed Central

Anderson, Nels C.

1969-01-01

These studies have developed and tested an experimental approach to the study of membrane ionic conductance mechanisms in strips of uterine smooth muscle. The experimental and theoretical basis for applying the double sucrose-gap technique is described along with the limitations of this system. Nonpropagating membrane action potentials were produced in response to depolarizing current pulses under current-clamp conditions. The stepwise change of membrane potential under voltage-clamp conditions resulted in a family of ionic currents with voltage- and time-dependent characteristics. In sodium-free solution the peak transient current decreased and its equilibrium potential shifted along the voltage axis toward a more negative internal potential. These studies indicate a sodium-dependent, regenerative excitation mechanism. PMID:5796366

Control of resting membrane potential by delayed rectifier potassium currents in ferret airway smooth muscle cells.

PubMed Central

Fleischmann, B K; Washabau, R J; Kotlikoff, M I

1993-01-01

1. In order to determine the physiological role of specific potassium currents in airway smooth muscle, potassium currents were measured in freshly dissociated ferret trachealis cells using the nystatin-permeabilized, whole-cell method, at 35 degrees C. 2. The magnitude of the outward currents was markedly increased as bath temperature was increased from 22 to 35 degrees C. This increase was primarily due to the increase in maximum potassium conductance (gK,max), although there was also a small leftward shift in the relationship between gK and voltage at higher temperatures. The maximum conductance and the kinetics of current activation and inactivation were also temperature dependent. At 35 degrees C, gating of the current was steeply voltage dependent between -40 and 0 mV. Current activation was well fitted by fourth-order kinetics; the mean time constants of activation (30 mV clamp step) were 1.09 +/- 0.17 and 1.96 +/- 0.27 ms at 35 and 22 degrees C, respectively. 3. Outward currents using the nystatin method were qualitatively similar to delayed rectifier currents recorded in dialysed cells with high calcium buffering capacity solutions. 4-Aminopyridine (4-AP; 2 mM), a specific blocker of delayed rectifier potassium channels in this tissue, inhibited over 80% of the outward current evoked by voltage-clamp steps to between -10 and +20 mV (n = 6). Less than 5% of the outward current was blocked over the same voltage range by charybdotoxin (100 nM; n = 15), a specific antagonist of large-conductance, calcium-activated potassium channels in this tissue. 4. The degree to which delayed rectifier and calcium-activated potassium conductances control resting membrane potential was examined in current-clamp experiments. The resting membrane potential of current clamped cells was -33.6 +/- 1.0 mV (n = 62). Application of 4-AP (2 mM) resulted in a 14.4 +/- 1.0 mV depolarization (n = 8) and an increase in input resistance. Charybdotoxin (100 nM) had no effect on resting membrane potential (n = 6). 5. Force measurements were made in isolated strips of trachealis muscle to determine the effect of pharmacological blockade of individual potassium conductances on resting tone. In the presence of tetrodotoxin (1 microM) and atropine (1 microM), 4-AP increased baseline tension in a dose-dependent manner, with an EC50 of 1.8 mM (n = 13); application of 5 mM 4-AP increased tone to 86.8 +/- 8.1% of that produced by 1 microM methacholine, and this tone was almost completely inhibited by nifedipine (1 microM).(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8271220

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.

Equilibrium potential for the postsynaptic response in the squid giant synapse.

PubMed

Llinás, R; Joyner, R W; Nicholson, C

1974-11-01

The reversal potential for the EPSP in the squid giant synapse has been studied by means of an intracellular, double oil gap technique. This method allows the electrical isolation of a portion of the axon from the rest of the fiber and generates a quasi-isopotential segment. In order to make the input resistance of this nerve segment as constant as possible, the electroresponsive properties of the nerve membrane were blocked by intracellular injection of tetraethylammonium (TEA) and local extracellular application of tetrodotoxin (TTX). Thus, EPSP's could be evoked in the isolated segment with a minimal amount of electroresponsive properties. The reversal potential for the EPSP (EEPSP) was measured by recording the synaptic potential or the synaptic current during voltage clamping. The results indicate that EEPSP may vary from +15 to +25 mV, which is more positive than would be expected for a 1:1 conductance change for Na(+) and K(+) (approximately -15 mV) and too negative for a pure Na(+) conductance ((+)40 mV). This latter value (E(Na)) was directly determined in the voltage clamp experiments. The results suggest that the synaptic potential is probably produced by a permeability change to Na(+) to K(+) in a 4:1 ratio. No change in time-course was observed in the synaptic current at clamp levels of -100 and +90 mV. The implications of a variable ratio for Na(+)-K(+) permeability in subsynaptic-postsynaptic membranes are discussed.

Enhanced excitability and down-regulated voltage-gated potassium channels in colonic drg neurons from neonatal maternal separation rats.

PubMed

Luo, Jia-Lie; Qin, Hong-Yan; Wong, Chun-Kit; Tsang, Suk-Ying; Huang, Yu; Bian, Zhao-Xiang

2011-05-01

Irritable bowel syndrome (IBS), characterized mainly by abdominal pain, is a functional bowel disorder. The present study aimed to examine changes in the excitability and the activity of the voltage-gated K(+) channel in dorsal root ganglia (DRG) neurons innervating the colon of rats subjected to neonatal maternal separation (NMS). Colonic DRG neurons from NMS rats as identified by FAST DiI™ labeling showed an increased cell size compared with those from nonhandled (NH) rats. Whole cell current-clamp recordings showed that colonic DRG neurons from NMS rats displayed: 1) depolarized resting membrane potential; 2) increased input resistance; 3) a dramatic reduction in rheobase; and 4) a significant increase in the number of action potentials evoked at twice rheobase. Whole cell voltage-clamp recordings revealed that neurons from both groups exhibited transient A-type (I(A)) and delayed rectifier (I(K)) K(+) currents. Compared with NH rat neurons, the averaged density of I(K) was significantly reduced in NMS rat neurons. Furthermore, the Kv1.2 expression was significantly decreased in NMS rat colonic DRG neurons. These results suggest that NMS increases the excitability of colonic DRG neurons mainly by suppressing the I(K) current, which is likely accounted for by the downregulation of the Kv1.2 expression and somal hypertrophy. This study demonstrates the alteration of delayed rectifier K current and Kv1.2 expression in DRG neurons from IBS model rats, representing a molecular mechanism underlying visceral pain and sensitization in IBS, suggesting the potential of Kv1.2 as a therapeutic target for the treatment of IBS. Copyright © 2011 American Pain Society. Published by Elsevier Inc. All rights reserved.

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

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

Measuring Feedforward Inhibition and Its Impact on Local Circuit Function.

PubMed

Hull, Court

2017-05-01

This protocol describes a series of approaches to measure feedforward inhibition in acute brain slices from the cerebellar cortex. Using whole-cell voltage and current clamp recordings from Purkinje cells in conjunction with electrical stimulation of the parallel fibers, these methods demonstrate how to measure the relationship between excitation and inhibition in a feedforward circuit. This protocol also describes how to measure the impact of feedforward inhibition on Purkinje cell excitability, with an emphasis on spike timing. © 2017 Cold Spring Harbor Laboratory Press.

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.

A contraction-related component of slow inward current in dog ventricular muscle and its relation to Na(+)-Ca2+ exchange.

PubMed Central

Simurda, J; Simurdová, M; Bravený, P; Sumbera, J

1992-01-01

1. The slow inward current component related to contraction (Isic) was studied in voltage clamp experiments on canine ventricular trabeculae at 30 degrees C with the aims of (a) estimating its relation to electrogenic Na(+)-Ca2+ exchange and (b) comparing it with similar currents as reported in cardiac myocytes. 2. Isic may be recorded under conditions of augmented contractility in response to depolarizing pulses below the threshold of the classic slow inward current (presumably mediated by L-type Ca2+ channels). In responses to identical depolarizing clamp pulses the peak value of Isic is directly related to the amplitude of contraction (Fmax). Isic peaks about 60 ms after the onset of depolarization and declines with a half-time of about 110 ms. 3. The voltage threshold of Isic activation is the same as the threshold of contraction. The positive inotropic clamp preconditions shift both thresholds to more negative values of membrane voltage, i.e. below the threshold of the classic slow inward current. 4. Isic may also be recorded as a slowly decaying inwardly directed current 'tail' after depolarizing pulses. In this representation the peak value of Isic changes with duration of the depolarizing pulses, again in parallel with Fmax. In response to pulses shorter than 100 ms both variables increase with depolarization time. If initial conditions remain constant, further prolongation of the pulse does not significantly influence either one (tail currents follow a common envelope). 5. Isic differs from classic slow inward current by: (a) its direct relation to contraction, (b) the slower decay of the current tail on repolarization, (c) slower restitution corresponding to the mechanical restitution, (d) its relative insensitivity to Ca(2+)-blocking agents (the decrease of Isic is secondary to the negative inotropic of Ca(2+)-blocking agents (the decrease of Isic is secondary to the negative inotropic effect) and (e) its disappearance after Sr2+ substitution for Ca2+. 6. The manifestations of Isic in multicellular preparations do not differ significantly from those reported in isolated myocytes (in contrast to calcium current). 7. The analysis of the correlation between Isic and Fmax transients during trains of identical test depolarizing pulses at variable extra- and intracellular ionic concentrations (changes of [Ca2+]o, 50% Li+ substitution for Na+, strophanthidin) indicate that the observed effects conform to the predictions based on a quantitative model of Na(+)-Ca2+ exchange. 8. It is concluded that Isic is activated by a transient increase of [Ca2+]i, in consequence of the release from the reticular stores.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1293284

A complete dc characterization of a constant-frequency, clamped-mode, series-resonant converter

NASA Technical Reports Server (NTRS)

Tsai, Fu-Sheng; Lee, Fred C.

1988-01-01

The dc behavior of a clamped-mode series-resonant converter is characterized systematically. Given a circuit operating condition, the converter's mode of operation is determined and various circuit parameters are calculated, such as average inductor current (load current), rms inductor current, peak capacitor voltage, rms switch currents, average diode currents, switch turn-on currents, and switch turn-off currents. Regions of operation are defined, and various circuit characteristics are derived to facilitate the converter design.

Block of voltage-gated potassium channels by Pacific ciguatoxin-1 contributes to increased neuronal excitability in rat sensory neurons

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

Birinyi-Strachan, Liesl C.; Gunning, Simon J.; Lewis, Richard J.

2005-04-15

The present study investigated the actions of the polyether marine toxin Pacific ciguatoxin-1 (P-CTX-1) on neuronal excitability in rat dorsal root ganglion (DRG) neurons using patch-clamp recording techniques. Under current-clamp conditions, bath application of 2-20 nM P-CTX-1 caused a rapid, concentration-dependent depolarization of the resting membrane potential in neurons expressing tetrodotoxin (TTX)-sensitive voltage-gated sodium (Na{sub v}) channels. This action was completely suppressed by the addition of 200 nM TTX to the external solution, indicating that this effect was mediated through TTX-sensitive Na{sub v} channels. In addition, P-CTX-1 also prolonged action potential and afterhyperpolarization (AHP) duration. In a subpopulation of neurons,more » P-CTX-1 also produced tonic action potential firing, an effect that was not accompanied by significant oscillation of the resting membrane potential. Conversely, in neurons expressing TTX-resistant Na{sub v} currents, P-CTX-1 failed to alter any parameter of neuronal excitability examined in this study. Under voltage-clamp conditions in rat DRG neurons, P-CTX-1 inhibited both delayed-rectifier and 'A-type' potassium currents in a dose-dependent manner, actions that occurred in the absence of alterations to the voltage dependence of activation. These actions appear to underlie the prolongation of the action potential and AHP, and contribute to repetitive firing. These data indicate that a block of potassium channels contributes to the increase in neuronal excitability, associated with a modulation of Na{sub v} channel gating, observed clinically in response to ciguatera poisoning.« less

Iberiotoxin-sensitive and -insensitive BK currents in Purkinje neuron somata

PubMed Central

Benton, Mark D.; Lewis, Amanda H.; Bant, Jason S.

2013-01-01

Purkinje cells have specialized intrinsic ionic conductances that generate high-frequency action potentials. Disruptions of their Ca or Ca-activated K (KCa) currents correlate with altered firing patterns in vitro and impaired motor behavior in vivo. To examine the properties of somatic KCa currents, we recorded voltage-clamped KCa currents in Purkinje cell bodies isolated from postnatal day 17–21 mouse cerebellum. Currents were evoked by endogenous Ca influx with approximately physiological Ca buffering. Purkinje somata expressed voltage-activated, Cd-sensitive KCa currents with iberiotoxin (IBTX)-sensitive (>100 nS) and IBTX-insensitive (>75 nS) components. IBTX-sensitive currents activated and partially inactivated within milliseconds. Rapid, incomplete macroscopic inactivation was also evident during 50- or 100-Hz trains of 1-ms depolarizations. In contrast, IBTX-insensitive currents activated more slowly and did not inactivate. These currents were insensitive to the small- and intermediate-conductance KCa channel blockers apamin, scyllatoxin, UCL1684, bicuculline methiodide, and TRAM-34, but were largely blocked by 1 mM tetraethylammonium. The underlying channels had single-channel conductances of ∼150 pS, suggesting that the currents are carried by IBTX-resistant (β4-containing) large-conductance KCa (BK) channels. IBTX-insensitive currents were nevertheless increased by small-conductance KCa channel agonists EBIO, chlorzoxazone, and CyPPA. During trains of brief depolarizations, IBTX-insensitive currents flowed during interstep intervals, and the accumulation of interstep outward current was enhanced by EBIO. In current clamp, EBIO slowed spiking, especially during depolarizing current injections. The two components of BK current in Purkinje somata likely contribute differently to spike repolarization and firing rate. Moreover, augmentation of BK current may partially underlie the action of EBIO and chlorzoxazone to alleviate disrupted Purkinje cell firing associated with genetic ataxias. PMID:23446695

Perturbed atrial calcium handling in an ovine model of heart failure: Potential roles for reductions in the L-type calcium current

PubMed Central

Clarke, Jessica D.; Caldwell, Jessica L.; Horn, Margaux A.; Bode, Elizabeth F.; Richards, Mark A.; Hall, Mark C.S.; Graham, Helen K.; Briston, Sarah J.; Greensmith, David J.; Eisner, David A.; Dibb, Katharine M.; Trafford, Andrew W.

2015-01-01

Heart failure (HF) is commonly associated with reduced cardiac output and an increased risk of atrial arrhythmias particularly during β-adrenergic stimulation. The aim of the present study was to determine how HF alters systolic Ca2 + and the response to β-adrenergic (β-AR) stimulation in atrial myocytes. HF was induced in sheep by ventricular tachypacing and changes in intracellular Ca2 + concentration studied in single left atrial myocytes under voltage and current clamp conditions. The following were all reduced in HF atrial myocytes; Ca2 + transient amplitude (by 46% in current clamped and 28% in voltage clamped cells), SR dependent rate of Ca2 + removal (kSR, by 32%), L-type Ca2 + current density (by 36%) and action potential duration (APD90 by 22%). However, in HF SR Ca2 + content was increased (by 19%) when measured under voltage-clamp stimulation. Inhibiting the L-type Ca2 + current (ICa-L) in control cells reproduced both the decrease in Ca2 + transient amplitude and increase of SR Ca2 + content observed in voltage-clamped HF cells. During β-AR stimulation Ca2 + transient amplitude was the same in control and HF cells. However, ICa-L remained less in HF than control cells whilst SR Ca2 + content was highest in HF cells during β-AR stimulation. The decrease in ICa-L that occurs in HF atrial myocytes appears to underpin the decreased Ca2 + transient amplitude and increased SR Ca2 + content observed in voltage-clamped cells. PMID:25463272

Prevention of Ca(2+)-mediated action potentials in GABAergic local circuit neurones of rat thalamus by a transient K+ current.

PubMed Central

Pape, H C; Budde, T; Mager, R; Kisvárday, Z F

1994-01-01

1. Neurones enzymatically dissociated from the rat dorsal lateral geniculate nucleus (LGN) were identified as GABAergic local circuit interneurones and geniculocortical relay cells, based upon quantitative analysis of soma profiles, immunohistochemical detection of GABA or glutamic acid decarboxylase, and basic electrogenic behaviour. 2. During whole-cell current-clamp recording, isolated LGN neurones generated firing patterns resembling those in intact tissue, with the most striking difference relating to the presence in relay cells of a Ca2+ action potential with a low threshold of activation, capable of triggering fast spikes, and the absence of a regenerative Ca2+ response with a low threshold of activation in local circuit cells. 3. Whole-cell voltage-clamp experiments demonstrated that both classes of LGN neurones possess at least two voltage-dependent membrane currents which operate in a range of membrane potentials negative to the threshold for generation of Na(+)-K(+)-mediated spikes: the T-type Ca2+ current (IT) and an A-type K+ current (IA). Taking into account the differences in membrane surface area, the average size of IT was similar in the two types of neurones, and interneurones possessed a slightly larger A-conductance. 4. In local circuit neurones, the ranges of steady-state inactivation and activation of IT and IA were largely overlapping (VH = 81.1 vs. -82.8 mV), both currents activated at around -70 mV, and they rapidly increased in amplitude with further depolarization. In relay cells, the inactivation curve of IT was negatively shifted along the voltage axis by about 20 mV compared with that of IA (Vh = -86.1 vs. -69.2 mV), and the activation threshold for IT (at -80 mV) was 20 mV more negative than that for IA. In interneurones, the activation range of IT was shifted to values more positive than that in relay cells (Vh = -54.9 vs. -64.5 mV), whereas the activation range of IA was more negative (Vh = -25.2 vs. -14.5 mV). 5. Under whole-cell voltage-clamp conditions that allowed the combined activation of Ca2+ and K+ currents, depolarizing voltage steps from -110 mV evoked inward currents resembling IT in relay cells and small outward currents indicative of IA in local circuit neurones. After blockade of IA with 4-aminopyridine (4-AP), the same pulse protocol produced IT in both types of neurones. Under current clamp, 4-AP unmasked a regenerative membrane depolarization with a low threshold of activation capable of triggering fast spikes in local circuit neurones.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 1 PMID:7965855

The action of alcohols and other non-ionic surface active substances on the sodium current of the squid giant axon.

PubMed Central

Haydon, D A; Urban, B W

1983-01-01

The effects of several n-alkanols and n-alkyl oxyethylene alcohols, methyl octanoate, glycerol 1-monooctanoate and dioctanoyl phosphatidylcholine on the ionic currents and electrical capacity of the squid giant axon membrane have been examined. The peak inward current in voltage-clamped axons was reduced reversibly by each substance. For n-pentanol to n-decanol the concentrations required to suppress the peak inward current by 50% were determined. From these data, it was estimated that the standard free energy per CH2 for adsorption to the site of action was -3.04 kJ mole-1, as compared with -3.11 kJ mole-1 for adsorption into phospholipid bilayers or an n-alkane/aqueous solution interface. The membrane capacity at 100 kHz was not greatly by any of the test substances at concentrations which reduced the inward current by 50%. Na currents under voltage clamp were recorded in intracellularly perfused axons before, during and sometimes after exposure to the test substances and the records were fitted with equations similar to those proposed by Hodgkin & Huxley (1952). Shifts in the curves of the steady-state activation and inactivation parameters (m infinity and h infinity) against membrane potential, changes in the peak heights of the activation and inactivation time constants (tau m and tau h) and reductions in the maximum Na conductance (gNa) have been tabulated. All of the test substances shifted the voltage dependence of the steady-state activation in the depolarizing direction and lowered the peak time constants for both activation and inactivation. The origins of these effects, and of the differences in the present results from those of the hydrocarbons (Haydon & Urban, 1983), have been discussed in terms of the physico-chemical properties of the two groups of substances and with reference to their effects on artificial membranes. PMID:6312030

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.

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.

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

Mechanisms underlying activation of transient BK current in rabbit urethral smooth muscle cells and its modulation by IP3-generating agonists

PubMed Central

Kyle, Barry D.; Bradley, Eamonn; Large, Roddy; Sergeant, Gerard P.; McHale, Noel G.; Thornbury, Keith D.

2013-01-01

We used the perforated patch-clamp technique at 37°C to investigate the mechanisms underlying the activation of a transient large-conductance K+ (tBK) current in rabbit urethral smooth muscle cells. The tBK current required an elevation of intracellular Ca2+, resulting from ryanodine receptor (RyR) activation via Ca2+-induced Ca2+ release, triggered by Ca2+ influx through L-type Ca2+ (CaV) channels. Carbachol inhibited tBK current by reducing Ca2+ influx and Ca2+ release and altered the shape of spike complexes recorded under current-clamp conditions. The tBK currents were blocked by iberiotoxin and penitrem A (300 and 100 nM, respectively) and were also inhibited when external Ca2+ was removed or the CaV channel inhibitors nifedipine (10 μM) and Cd2+ (100 μM) were applied. The tBK current was inhibited by caffeine (10 mM), ryanodine (30 μM), and tetracaine (100 μM), suggesting that RyR-mediated Ca2+ release contributed to the activation of the tBK current. When IP3 receptors (IP3Rs) were blocked with 2-aminoethoxydiphenyl borate (2-APB, 100 μM), the amplitude of the tBK current was not reduced. However, when Ca2+ release via IP3Rs was evoked with phenylephrine (1 μM) or carbachol (1 μM), the tBK current was inhibited. The effect of carbachol was abolished when IP3Rs were blocked with 2-APB or by inhibition of muscarinic receptors with the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (1 μM). Under current-clamp conditions, bursts of action potentials could be evoked with depolarizing current injection. Carbachol reduced the number and amplitude of spikes in each burst, and these effects were reduced in the presence of 2-APB. In the presence of ryanodine, the number and amplitude of spikes were also reduced, and carbachol was without further effect. These data suggest that IP3-generating agonists can modulate the electrical activity of rabbit urethral smooth muscle cells and may contribute to the effects of neurotransmitters on urethral tone. PMID:23804200

Zonal variations in K+ currents in vestibular crista calyx terminals

PubMed Central

Meredith, Frances L.

2014-01-01

We developed a rodent crista slice to investigate regional variations in electrophysiological properties of vestibular afferent terminals. Thin transverse slices of the gerbil crista ampullaris were made and electrical properties of calyx terminals in central zones (CZ) and peripheral zones (PZ) compared with whole cell patch clamp. Spontaneous action potential firing was observed in 25% of current-clamp recordings and was either regular or irregular in both zones. Firing was abolished when extracellular choline replaced Na+ but persisted when hair cell mechanotransduction channels or calyx AMPA receptors were blocked. This suggests that ion channels intrinsic to the calyx can generate spontaneous firing. In response to depolarizing voltage steps, outward K+ currents were observed at potentials above −60 mV. K+ currents in PZ calyces showed significantly more inactivation than currents in CZ calyces. Underlying K+ channel populations contributing to these differences were investigated. The KCNQ channel blocker XE991 dihydrochloride blocked a slowly activating, sustained outward current in both PZ and CZ calyces, indicating the presence of KCNQ channels. Mean reduction was greatest in PZ calyces. XE991 also reduced action potential firing frequency in CZ and PZ calyces and broadened mean action potential width. The K+ channel blocker 4-aminopyridine (10–50 μM) blocked rapidly activating, moderately inactivating currents that were more prevalent in PZ calyces. α-Dendrotoxin, a selective blocker of KV1 channels, reduced outward currents in CZ calyces but not in PZ calyces. Regional variations in K+ conductances may contribute to different firing responses in calyx afferents. PMID:25343781

Zonal variations in K+ currents in vestibular crista calyx terminals.

PubMed

Meredith, Frances L; Rennie, Katherine J

2015-01-01

We developed a rodent crista slice to investigate regional variations in electrophysiological properties of vestibular afferent terminals. Thin transverse slices of the gerbil crista ampullaris were made and electrical properties of calyx terminals in central zones (CZ) and peripheral zones (PZ) compared with whole cell patch clamp. Spontaneous action potential firing was observed in 25% of current-clamp recordings and was either regular or irregular in both zones. Firing was abolished when extracellular choline replaced Na(+) but persisted when hair cell mechanotransduction channels or calyx AMPA receptors were blocked. This suggests that ion channels intrinsic to the calyx can generate spontaneous firing. In response to depolarizing voltage steps, outward K(+) currents were observed at potentials above -60 mV. K(+) currents in PZ calyces showed significantly more inactivation than currents in CZ calyces. Underlying K(+) channel populations contributing to these differences were investigated. The KCNQ channel blocker XE991 dihydrochloride blocked a slowly activating, sustained outward current in both PZ and CZ calyces, indicating the presence of KCNQ channels. Mean reduction was greatest in PZ calyces. XE991 also reduced action potential firing frequency in CZ and PZ calyces and broadened mean action potential width. The K(+) channel blocker 4-aminopyridine (10-50 μM) blocked rapidly activating, moderately inactivating currents that were more prevalent in PZ calyces. α-Dendrotoxin, a selective blocker of KV1 channels, reduced outward currents in CZ calyces but not in PZ calyces. Regional variations in K(+) conductances may contribute to different firing responses in calyx afferents. Copyright © 2015 the American Physiological Society.

Multi-neuron intracellular recording in vivo via interacting autopatching robots

PubMed Central

Holst, Gregory L; Singer, Annabelle C; Han, Xue; Brown, Emery N

2018-01-01

The activities of groups of neurons in a circuit or brain region are important for neuronal computations that contribute to behaviors and disease states. Traditional extracellular recordings have been powerful and scalable, but much less is known about the intracellular processes that lead to spiking activity. We present a robotic system, the multipatcher, capable of automatically obtaining blind whole-cell patch clamp recordings from multiple neurons simultaneously. The multipatcher significantly extends automated patch clamping, or 'autopatching’, to guide four interacting electrodes in a coordinated fashion, avoiding mechanical coupling in the brain. We demonstrate its performance in the cortex of anesthetized and awake mice. A multipatcher with four electrodes took an average of 10 min to obtain dual or triple recordings in 29% of trials in anesthetized mice, and in 18% of the trials in awake mice, thus illustrating practical yield and throughput to obtain multiple, simultaneous whole-cell recordings in vivo. PMID:29297466

Parallel multipoint recording of aligned and cultured neurons on corresponding Micro Channel Array toward on-chip cell analysis.

PubMed

Tonomura, W; Moriguchi, H; Jimbo, Y; Konishi, S

2008-01-01

This paper describes an advanced Micro Channel Array (MCA) so as to record neuronal network at multiple points simultaneously. Developed MCA is designed for neuronal network analysis which has been studied by co-authors using MEA (Micro Electrode Arrays) system. The MCA employs the principle of the extracellular recording. Presented MCA has the following advantages. First of all, the electrodes integrated around individual micro channels are electrically isolated for parallel multipoint recording. Sucking and clamping of cells through micro channels is expected to improve the cellular selectivity and S/N ratio. In this study, hippocampal neurons were cultured on the developed MCA. As a result, the spontaneous and evoked spike potential could be recorded by sucking and clamping the cells at multiple points. Herein, we describe the successful experimental results together with the design and fabrication of the advanced MCA toward on-chip analysis of neuronal network.

Calcium Currents of Olfactory Bulb Juxtaglomerular Cells: Profile and Multiple Conductance Plateau Potential Simulation

PubMed Central

Masurkar, Arjun V.; Chen, Wei R.

2011-01-01

The olfactory glomerulus is the locus of information transfer between olfactory sensory neurons and output neurons of the olfactory bulb. Juxtaglomerular cells (JGCs) may influence intraglomerular processing by firing plateau potentials that support multiple spikes. It is unclear what inward currents mediate this firing pattern. In previous work, we characterized potassium currents of JGCs. We focus here on the inward currents using whole cell current clamp and voltage recording in a rat in vitro slice preparation, as well as computer simulation. We first showed that sodium current was not required to mediate plateau potentials. Voltage clamp characterization of calcium current (ICa) determined that ICa consisted of a slow activating, rapidly inactivating (τ10%–90% rise 6–8ms, τinactivation 38–77ms) component Icat1, similar to T-type currents, and a sustained (τinactivation≫500ms) component Icat2, likely composed of L-type and P/Q-type currents. We used computer simulation to test their roles in plateau potential firing. We robustly modeled Icat1 and Icat2 to Hodgkin-Huxley schemes (m3h and m2, respectively) and simulated a JGC plateau potential with 6 conductances: calcium currents as above, potassium currents from our prior study (A-type Ikt1, D-type Ikt2, delayed rectifier Ikt3), and a fast sodium current (INa). We demonstrated that Icat1 was required for mediating the plateau potential, unlike INa and Icat2, and its τinactivation determined plateau duration. We also found that Ikt1 dictated plateau potential shape more than Ikt2 and Ikt3. The influence of these two transient and opposing conductances suggests a unique mechanism of plateau potential physiology. PMID:21704681

Dynamic, nonlinear feedback regulation of slow pacemaking by A-type potassium current in ventral tegmental area neurons.

PubMed

Khaliq, Zayd M; Bean, Bruce P

2008-10-22

We analyzed ionic currents that regulate pacemaking in dopaminergic neurons of the mouse ventral tegmental area by comparing voltage trajectories during spontaneous firing with ramp-evoked currents in voltage clamp. Most recordings were made in brain slice, with key experiments repeated using acutely dissociated neurons, which gave identical results. During spontaneous firing, net ionic current flowing between spikes was calculated from the time derivative of voltage multiplied by cell capacitance, signal-averaged over many firing cycles to enhance resolution. Net inward interspike current had a distinctive nonmonotonic shape, reaching a minimum (generally <1 pA) between -60 and -55 mV. Under voltage clamp, ramps over subthreshold voltages elicited a time- and voltage-dependent outward current that peaked near -55 mV. This current was undetectable with 5 mV/s ramps and increased steeply with depolarization rate over the range (10-50 mV/s) typical of natural pacemaking. Ramp-evoked subthreshold current was resistant to alpha-dendrotoxin, paxilline, apamin, and tetraethylammonium but sensitive to 4-aminopyridine and 0.5 mM Ba2+, consistent with A-type potassium current (I(A)). Same-cell comparison of currents elicited by various ramp speeds with natural spontaneous depolarization showed how the steep dependence of I(A) on depolarization rate results in small net inward currents during pacemaking. These results reveal a mechanism in which subthreshold I(A) is near zero at steady state, but is engaged at depolarization rates >10 mV/s to act as a powerful, supralinear feedback element. This feedback mechanism explains how net ionic current can be constrained to <1-2 pA but reliably inward, thus enabling slow, regular firing.

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.

Dendritic calcium channels and their activation by synaptic signals in auditory coincidence detector neurons.

PubMed

Blackmer, Trillium; Kuo, Sidney P; Bender, Kevin J; Apostolides, Pierre F; Trussell, Laurence O

2009-08-01

The avian nucleus laminaris (NL) encodes the azimuthal location of low-frequency sound sources by detecting the coincidence of binaural signals. Accurate coincidence detection requires precise developmental regulation of the lengths of the fine, bitufted dendrites that characterize neurons in NL. Such regulation has been suggested to be driven by local, synaptically mediated, dendritic signals such as Ca(2+). We examined Ca(2+) signaling through patch clamp and ion imaging experiments in slices containing nucleus laminaris from embryonic chicks. Voltage-clamp recordings of neurons located in the NL showed the presence of large Ca(2+) currents of two types, a low voltage-activated, fast inactivating Ni(2+) sensitive channel resembling mammalian T-type channels, and a high voltage-activated, slowly inactivating Cd(2+) sensitive channel. Two-photon Ca(2+) imaging showed that both channel types were concentrated on dendrites, even at their distal tips. Single action potentials triggered synaptically or by somatic current injection immediately elevated Ca(2+) throughout the entire cell. Ca(2+) signals triggered by subthreshold synaptic activity were highly localized. Thus when electrical activity is suprathreshold, Ca(2+) channels ensure that Ca(2+) rises in all dendrites, even those that are synaptically inactive.

A Comparison of the Performance and Application Differences Between Manual and Automated Patch-Clamp Techniques

PubMed Central

Yajuan, Xiao; Xin, Liang; Zhiyuan, Li

2012-01-01

The patch clamp technique is commonly used in electrophysiological experiments and offers direct insight into ion channel properties through the characterization of ion channel activity. This technique can be used to elucidate the interaction between a drug and a specific ion channel at different conformational states to understand the ion channel modulators’ mechanisms. The patch clamp technique is regarded as a gold standard for ion channel research; however, it suffers from low throughput and high personnel costs. In the last decade, the development of several automated electrophysiology platforms has greatly increased the screen throughput of whole cell electrophysiological recordings. New advancements in the automated patch clamp systems have aimed to provide high data quality, high content, and high throughput. However, due to the limitations noted above, automated patch clamp systems are not capable of replacing manual patch clamp systems in ion channel research. While automated patch clamp systems are useful for screening large amounts of compounds in cell lines that stably express high levels of ion channels, the manual patch clamp technique is still necessary for studying ion channel properties in some research areas and for specific cell types, including primary cells that have mixed cell types and differentiated cells that derive from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Therefore, further improvements in flexibility with regard to cell types and data quality will broaden the applications of the automated patch clamp systems in both academia and industry. PMID:23346269

2D and 3D assessment of sustentaculum tali screw fixation with or without Screw Targeting Clamp.

PubMed

De Boer, A Siebe; Van Lieshout, Esther M M; Vellekoop, Leonie; Knops, Simon P; Kleinrensink, Gert-Jan; Verhofstad, Michael H J

2017-12-01

Precise placement of sustentaculum tali screw(s) is essential for restoring anatomy and biomechanical stability of the calcaneus. This can be challenging due to the small target area and presence of neurovascular structures on the medial side. The aim was to evaluate the precision of positioning of the subchondral posterior facet screw and processus anterior calcanei screw with or without a Screw Targeting Clamp. The secondary aim was to evaluate the added value of peroperative 3D imaging over 2D radiographs alone. Twenty Anubifix™ embalmed, human anatomic lower limb specimens were used. A subchondral posterior facet screw and a processus anterior calcanei screw were placed using an extended lateral approach. A senior orthopedic trauma surgeon experienced in calcaneal fracture surgery and a senior resident with limited experience in calcaneal surgery performed screw fixation in five specimens with and in five specimens without the clamp. 2D lateral and axial radiographs and a 3D recording were obtained postoperatively. Anatomical dissection was performed postoperatively as a diagnostic golden standard in order to obtain the factual screw positions. Blinded assessment of quality of fixation was performed by two surgeons. In 2D, eight screws were considered malpositioned when placed with the targeting device versus nine placed freehand. In 3D recordings, two additional screws were malpositioned in each group as compared to the golden standard. As opposed to the senior surgeon, the senior resident seemed to get the best results using the Screw Targeting Clamp (number of malpositioned screws using freehand was eight, and using the targeting clamp five). In nine out of 20 specimens 3D images provided additional information concerning target area and intra-articular placement. Based on the 3D assessment, five additional screws would have required repositioning. Except for one, all screw positions were rated equally after dissection when compared with 3D examinations. This study does not show a substantial benefit between the Screw Targeting Clamp and the freehand technique as well between experienced and inexperienced surgeons. Data suggest that the clamp might help positioning sustentaculum tali screws, especially for inexperienced surgeons. Perioperative 3D recordings facilitate identification of malpositioned screws. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

PKCepsilon-dependent potentiation of TTX-resistant Nav1.8 current by neurokinin-1 receptor activation in rat dorsal root ganglion neurons.

PubMed

Cang, Chun-Lei; Zhang, Hua; Zhang, Yu-Qiu; Zhao, Zhi-Qi

2009-06-30

Substance P (SP), which mainly exists in a subtype of small-diameter dorsal root ganglion (DRG) neurons, is an important signal molecule in pain processing in the spinal cord. Our previous results have proved the expression of SP receptor neurokinin-1 (NK-1) on DRG neurons and its interaction with transient receptor potential vanilloid 1 (TRPV1) receptor. In this study we investigated the effect of NK-1 receptor agonist on Na(v)1.8, a tetrodotoxin (TTX)-resistant sodium channel, in rat small-diameter DRG neurons employing whole-cell patch clamp recordings. NK-1 agonist [Sar(9), Met(O2)(11)]-substance P (Sar-SP) significantly enhanced the Na(v)1.8 currents in a subgroup of small-diameter DRG neurons under both the normal and inflammatory situation, and the enhancement was blocked by NK-1 antagonist Win51708 and protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM), but not the protein kinase A (PKA) inhibitor H89. In particular, the inhibitor of PKCepsilon, a PKC isoform, completely blocked this effect. Under current clamp model, Sar-SP reduced the amount of current required to evoke action potentials and increased the firing rate in a subgroup of DRG neurons. These data suggest that activation of NK-1 receptor potentiates Na(v)1.8 sodium current via PKCepsilon-dependent signaling pathway, probably participating in the generation of inflammatory hyperalgesia.

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.

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.

Effects of L-glutamate on 1F Helix aspersa neurons

NASA Astrophysics Data System (ADS)

Bernal-Martínez, Juan; Ortega Soto, Arturo

2004-09-01

The aim of this work is to characterize the effect of L-glut and related compounds on the electrical properties of 1F identified neurons of the garden snail Helix aspersa. We used intracellular recording experiments with regular microelectrodes, in current clamp conditions. We report here that the putative L-glut receptor present in 1F Helix neurons has some similarities with the L-glut receptor present in vertebrates, regarding ionic permeability and biophysical properties. However, these responses show different pharmacological properties from those receptors found in vertebrates and mammals.

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.

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.

Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X).

PubMed

Thomas, Dierk; Hammerling, Bettina C; Wimmer, Anna-Britt; Wu, Kezhong; Ficker, Eckhard; Kuryshev, Yuri A; Scherer, Daniel; Kiehn, Johann; Katus, Hugo A; Schoels, Wolfgang; Karle, Christoph A

2004-12-01

The human ether-a-go-go-related gene (hERG) encodes the rapid component of the cardiac repolarizing delayed rectifier potassium current, I(Kr). The direct interaction of the commonly used protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM I) with hERG, KvLQT1/minK, and I(Kr) currents was investigated in this study. hERG and KvLQT1/minK channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. In addition, hERG currents in stably transfected human embryonic kidney (HEK 293) cells, native I(Kr) currents and action potentials in isolated guinea pig ventricular cardiomyocytes were recorded using whole-cell patch clamp electrophysiology. Bisindolylmaleimide I blocked hERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner with IC(50) values of 1.0 and 13.2 muM, respectively. hERG channels were primarily blocked in the open state in a frequency-independent manner. Analysis of the voltage-dependence of block revealed a reduction of inhibition at positive membrane potentials. BIM I caused a shift of -20.3 mV in the voltage-dependence of inactivation. The point mutations tyrosine 652 alanine (Y652A) and phenylalanine 656 alanine (F656A) attenuated hERG current blockade, indicating that BIM I binds to a common drug receptor within the pore region. KvLQT1/minK currents were not significantly altered by BIM I. Finally, 1 muM BIM I reduced native I(Kr) currents by 69.2% and lead to action potential prolongation. In summary, PKC-independent effects have to be carefully considered when using BIM I as PKC inhibitor in experimental models involving hERG channels and I(Kr) currents.

Effect of urinary trypsin inhibitor on potassium currents: fetus modulates membrane excitability by production of UTI.

PubMed

Takeuchi, Kinya; Fukuda, Atsuo; Kanayama, Naohiro

2004-01-01

Amniotic fluid contains a significant level of urinary trypsin inhibitor (UTI). Previously, we reported that UTI inhibits calcium influx of myometrium and it is effective in preventing uterine contraction. This study examined the effects of UTI upon potassium channels, which is important for membrane excitability. Whole-cell patch-clamp recordings were performed in fibroblasts derived from human fetal skin. Potassium currents were recorded and the effects of exogenous UTI and/or cadmium determined. Tetraethylammonium sensitive potassium currents were elicited by step or ramp stimulations at depolarized membrane potentials (over +30 mV). Administration of 1 micro M UTI significantly increased these potassium currents by 16.9%. When calcium channels were blocked by the administration of cadmium, UTI increased the rest of the potassium currents by 4.8%. This indicates that UTI increased calcium-dependent potassium currents by 94.8% but only increased voltage-dependent potassium currents by 4.8%. Urinary trypsin inhibitor is a physiological substance of fetal origin that modulates calcium-dependent and voltage-dependent potassium channels. These data suggest that UTI is capable of regulating the membrane properties of the fetal and myometrial cells in contact with amniotic fluid.

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.

Effect of complete hilar versus only renal artery clamping on renal histomorphology following ischemia/reperfusion injury in an experimental model.

PubMed

Umul, M; Cal, A C; Turna, B; Oktem, G; Aydın, H H

2016-01-01

To evaluate the effect of temporary complete hilar versus only renal artery clamping with different duration of warm ischemia on renal functions, and possibly identify a "safe" clamping type and duration of renal ischemia. Fifty male rabbits have been incorporated to study. Rabbits were subjected to ischemia/reperfusion injury by temporary vascular clamping. Reagents were randomized to 3 experimental groups (only renal artery clamping, complete hilar clamping, sham surgery) and sub-groups were determined according to different clamping times (30 and 60 minutes). Median laparotomy and left renal hilus dissection were performed to sham group. Only artery or complete hilar clamping was performed for 30 or 60 minutes by microvascular bulldog clamps to other reagents. Rabbits were sacrificed 10 days after primary surgery and left nephrectomy performed. Nephrectomy materials were evaluated for the level of nitric-oxide synthase (NOS) immunoreactivity, malondialdehyde (MDA) level and superoxide dismutase (SOD) activity and an electron microscopic examination was performed. NOS immunoreactivity was correlated with the temporary clamping time. We also observed that complete hilar vascular clamping entails an increase on NOS immunoreactivity. MDA levels were similar for all experimental surgery groups (p = 0.42). The SOD activity was decreased among all subgroups compared with sham surgery. But the significant decrease occurred in 30 minutes only artery and 30 minutes complete hilar clamping groups in proportion to sham surgery (p = 0.026 and p = 0.019, respectively). This current study suggested that only renal artery clamping under 30 minutes is more appropriate during renal surgical procedures requiring temporary vascular clamping.

Cardiomyocyte dysfunction during the chronic phase of Chagas disease.

PubMed

Roman-Campos, Danilo; Sales-Júnior, Policarpo; Duarte, Hugo Leonardo; Gomes, Eneas Ricardo; Guatimosim, Silvia; Ropert, Catherine; Gazzinelli, Ricardo Tostes; Cruz, Jader Santos

2013-04-01

Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of heart failure. We investigated modifications in the cellular electrophysiological and calcium-handling characteristics of an infected mouse heart during the chronic phase of the disease. The patch-clamp technique was used to record action potentials (APs) and L-type Ca2+ and transient outward K+ currents. [Ca2+]i changes were determined using confocal microscopy. Infected ventricular cells showed prolonged APs, reduced transient outward K+ and L-type Ca2+ currents and reduced Ca2+ release from the sarcoplasmic reticulum. Thus, the chronic phase of Chagas disease is characterised by cardiomyocyte dysfunction, which could lead to heart failure.

Cardiomyocyte dysfunction during the chronic phase of Chagas disease

PubMed Central

Roman-Campos, Danilo; Sales-Júnior, Policarpo; Duarte, Hugo Leonardo; Gomes, Eneas Ricardo; Guatimosim, Silvia; Ropert, Catherine; Gazzinelli, Ricardo Tostes; Cruz, Jader Santos

2013-01-01

Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of heart failure. We investigated modifications in the cellular electrophysiological and calcium-handling characteristics of an infected mouse heart during the chronic phase of the disease. The patch-clamp technique was used to record action potentials (APs) and L-type Ca2+ and transient outward K+ currents. [Ca2+]i changes were determined using confocal microscopy. Infected ventricular cells showed prolonged APs, reduced transient outward K+ and L-type Ca2+ currents and reduced Ca2+ release from the sarcoplasmic reticulum. Thus, the chronic phase of Chagas disease is characterised by cardiomyocyte dysfunction, which could lead to heart failure. PMID:23579807

Parallel multipoint recording of aligned and cultured neurons on micro channel array toward cellular network analysis.

PubMed

Tonomura, Wataru; Moriguchi, Hiroyuki; Jimbo, Yasuhiko; Konishi, Satoshi

2010-08-01

This paper describes an advanced Micro Channel Array (MCA) for recording electrophysiological signals of neuronal networks at multiple points simultaneously. The developed MCA is designed for neuronal network analysis which has been studied by the co-authors using the Micro Electrode Arrays (MEA) system, and employs the principles of extracellular recordings. A prerequisite for extracellular recordings with good signal-to-noise ratio is a tight contact between cells and electrodes. The MCA described herein has the following advantages. The electrodes integrated around individual micro channels are electrically isolated to enable parallel multipoint recording. Reliable clamping of a targeted cell through micro channels is expected to improve the cellular selectivity and the attachment between the cell and the electrode toward steady electrophysiological recordings. We cultured hippocampal neurons on the developed MCA. As a result, the spontaneous and evoked spike potentials could be recorded by sucking and clamping the cells at multiple points. In this paper, we describe the design and fabrication of the MCA and the successful electrophysiological recordings leading to the development of an effective cellular network analysis device.

Sumatriptan Inhibits TRPV1 Channels in Trigeminal Neurons

PubMed Central

Evans, M. Steven; Cheng, Xiangying; Jeffry, Joseph A.; Disney, Kimberly E.; Premkumar, Louis S.

2011-01-01

Objective To understand a possible role for transient potential receptor vanilloid 1 (TRPV1) ion channels in sumatriptan relief of pain mediated by trigeminal nociceptors. Background TRPV1 channels are expressed in small nociceptive sensory neurons. In dorsal root ganglia (DRG), TRPV1-containing nociceptors mediate certain types of inflammatory pain. Neurogenic inflammation of cerebral dura and blood vessels in the trigeminal nociceptive system is thought to be important in migraine pain, but the ion channels important in transducing migraine pain are not known. Sumatriptan is an agent effective in treatment of migraine and cluster headache. We hypothesized that sumatriptan might modulate activity of TRPV1 channels found in the trigeminal nociceptive system. Methods We used immunohistochemistry to detect the presence of TRPV1 channel protein, whole cell recording in acutely dissociated trigeminal ganglia (TG) to detect functionality of TRPV1 channels, and whole cell recording in trigeminal nucleus caudalis (TNC) to detect effects on release of neurotransmitters from trigeminal neurons onto second order sensory neurons. Effects specifically on TG neurons that project to cerebral dura were assessed by labeling dural nociceptors with DiI. Results Immunohistochemistry demonstrated that TRPV1 channels are present in cerebral dura, trigeminal ganglion, and in the trigeminal nucleus caudalis. Capsaicin, a TRPV1 agonist, produced depolarization and repetitive action potential firing in current clamp recordings and large inward currents in voltage clamp recordings from acutely dissociated TG neurons, demonstrating that TRPV1 channels are functional in trigeminal neurons. Capsaicin increased spontaneous excitatory postsynaptic currents (sEPSCs) in neurons of layer II in TNC slices, showing that these channels have a physiological effect on central synaptic transmission. Sumatriptan (10 μM), a selective anti-migraine drug inhibited TRPV1-mediated inward currents in TG. and capsaicin-elicited sEPSCs in TNC slices. The same effects of capsaicin and sumatriptan were found in acutely dissociated DiI-labeled TG neurons innervating cerebral dura. Conclusion Our results build on previous work indicating that TRPV1 channels in trigeminal nociceptors play a role in craniofacial pain. Our findings that TRPV1 is inhibited by the specific antimigraine drug sumatriptan, and that TRPV1 channels are functional in neurons projecting to cerebral dura suggests a specific role for these channels in migraine or cluster headache. PMID:22289052

ATP-induced current in isolated outer hair cells of guinea pig cochlea.

PubMed

Nakagawa, T; Akaike, N; Kimitsuki, T; Komune, S; Arima, T

1990-05-01

1. Electrical and pharmacologic properties of ATP-induced current in outer hair cells isolated from guinea pig cochlea were investigated in the whole-cell recording mode by the use of a conventional patch-clamp technique. 2. Under current-clamp conditions, rapid application of ATP depolarized the outer hair cells resulting in an increase in conductance. The ATP-induced response did not show any desensitization during a continuous application. 3. At a holding potential of -70 mV, the ATP-induced inward current increased in a sigmoidal fashion over the concentration range between 3 microM and 1 mM. The half-maximum concentration (EC50) was 12 microM and the Hill coefficient was 0.93. 4. The ATP-induced current had a reversal potential near 6 mV, which was close to the theoretical value (1 mV) calculated from the Goldman-Hodgkin-Katz equation for permeable intra- and extracellular cations. 5. In the current-voltage (I-V) relationship for the ATP response, a slight inward-going rectification was observed at more positive potentials than the reversal potential. 6. The substitution of extracellular Na+ by equimolar choline+ shifted the reversal potential of the ATP-induced current to more negative values. The substitution of Cs+ in the internal solution by N-methyl-D-glucamine+ (NMG+) shifted it in the positive direction. The reversal potential of ATP-induced current was also shifted to positive values with increasing extracellular Ca2+ concentration. A decrease of intracellular Cl- by gluconate- did not affect the reversal potential, thereby indicating that the ATP-induced current is carried through a large cation channel.(ABSTRACT TRUNCATED AT 250 WORDS)

The ionic bases of the action potential in isolated mouse cardiac Purkinje cell.

PubMed

Vaidyanathan, Ravi; O'Connell, Ryan P; Deo, Makarand; Milstein, Michelle L; Furspan, Philip; Herron, Todd J; Pandit, Sandeep V; Musa, Hassan; Berenfeld, Omer; Jalife, José; Anumonwo, Justus M B

2013-01-01

Collecting electrophysiological and molecular data from the murine conduction system presents technical challenges. Thus, only little advantage has been taken of numerous genetically engineered murine models to study excitation through the cardiac conduction system of the mouse. To develop an approach for isolating murine cardiac Purkinje cells (PCs), to characterize major ionic currents and to use the data to simulate action potentials (APs) recorded from PCs. Light microscopy was used to isolate and identify PCs from apical and septal cells. Current and voltage clamp techniques were used to record APs and whole cell currents. We then simulated a PC AP on the basis of our experimental data. APs recorded from PCs were significantly longer than those recorded from ventricular cells. The prominent plateau phase of the PC AP was very negative (≈-40 mV). Spontaneous activity was observed only in PCs. The inward rectifier current demonstrated no significant differences compared to ventricular myocytes (VMs). However, sodium current density was larger, and the voltage-gated potassium current density was significantly less in PCs compared with myocytes. T-type Ca(2+) currents (I(Ca,T)) were present in PCs but not VMs. Computer simulations suggest that I(Ca,T) and cytosolic calcium diffusion significantly modulate AP profile recorded in PCs, as compared to VMs. Our study provides the first comprehensive ionic profile of murine PCs. The data show unique features of PC ionic mechanisms that govern its excitation process. Experimental data and numerical modeling results suggest that a smaller voltage-gated potassium current and the presence of I(Ca,T) are important determinants of the longer and relatively negative plateau phase of the APs. Copyright © 2013 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Clinical aspects of incorporating cord clamping into stabilisation of preterm infants.

PubMed

Knol, Ronny; Brouwer, Emma; Vernooij, Alex S N; Klumper, Frans J C M; DeKoninck, Philip; Hooper, Stuart B; Te Pas, Arjan B

2018-04-21

Fetal to neonatal transition is characterised by major pulmonary and haemodynamic changes occurring in a short period of time. In the international neonatal resuscitation guidelines, comprehensive recommendations are available on supporting pulmonary transition and delaying clamping of the cord in preterm infants. Recent experimental studies demonstrated that the pulmonary and haemodynamic transition are intimately linked, could influence each other and that the timing of umbilical cord clamping should be incorporated into the respiratory stabilisation. We reviewed the current knowledge on how to incorporate cord clamping into stabilisation of preterm infants and the physiological-based cord clamping (PBCC) approach, with the infant's transitional status as key determinant of timing of cord clamping. This approach could result in optimal timing of cord clamping and has the potential to reduce major morbidities and mortality in preterm infants. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Functional Interaction between the Scaffold Protein Kidins220/ARMS and Neuronal Voltage-Gated Na+ Channels.

PubMed

Cesca, Fabrizia; Satapathy, Annyesha; Ferrea, Enrico; Nieus, Thierry; Benfenati, Fabio; Scholz-Starke, Joachim

2015-07-17

Kidins220 (kinase D-interacting substrate of 220 kDa)/ankyrin repeat-rich membrane spanning (ARMS) acts as a signaling platform at the plasma membrane and is implicated in a multitude of neuronal functions, including the control of neuronal activity. Here, we used the Kidins220(-/-) mouse model to study the effects of Kidins220 ablation on neuronal excitability. Multielectrode array recordings showed reduced evoked spiking activity in Kidins220(-/-) hippocampal networks, which was compatible with the increased excitability of GABAergic neurons determined by current-clamp recordings. Spike waveform analysis further indicated an increased sodium conductance in this neuronal subpopulation. Kidins220 association with brain voltage-gated sodium channels was shown by co-immunoprecipitation experiments and Na(+) current recordings in transfected HEK293 cells, which revealed dramatic alterations of kinetics and voltage dependence. Finally, an in silico interneuronal model incorporating the Kidins220-induced Na(+) current alterations reproduced the firing phenotype observed in Kidins220(-/-) neurons. These results identify Kidins220 as a novel modulator of Nav channel activity, broadening our understanding of the molecular mechanisms regulating network excitability. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Antagonism of Lidocaine Inhibition by Open-Channel Blockers That Generate Resurgent Na Current

PubMed Central

Bant, Jason S.; Aman, Teresa K.; Raman, Indira M.

2013-01-01

Na channels that generate resurgent current express an intracellular endogenous open-channel blocking protein, whose rapid binding upon depolarization and unbinding upon repolarization minimizes fast and slow inactivation. Na channels also bind exogenous compounds, such as lidocaine, which functionally stabilize inactivation. Like the endogenous blocking protein, these use-dependent inhibitors bind most effectively at depolarized potentials, raising the question of how lidocaine-like compounds affect neurons with resurgent Na current. We therefore recorded lidocaine inhibition of voltage-clamped, tetrodotoxin-sensitive Na currents in mouse Purkinje neurons, which express a native blocking protein, and in mouse hippocampal CA3 pyramidal neurons with and without a peptide from the cytoplasmic tail of NaVβ4 (the β4 peptide), which mimics endogenous open-channel block. To control channel states during drug exposure, lidocaine was applied with rapid-solution exchange techniques during steps to specific voltages. Inhibition of Na currents by lidocaine was diminished by either the β4 peptide or the native blocking protein. In peptide-free CA3 cells, prolonging channel opening with a site-3 toxin, anemone toxin II, reduced lidocaine inhibition; this effect was largely occluded by open-channel blockers, suggesting that lidocaine binding is favored by inactivation but prevented by open-channel block. In constant 100 μM lidocaine, current-clamped Purkinje cells continued to fire spontaneously. Similarly, the β4 peptide reduced lidocaine-dependent suppression of spiking in CA3 neurons in slices. Thus, the open-channel blocking protein responsible for resurgent current acts as a natural antagonist of lidocaine. Neurons with resurgent current may therefore be less susceptible to use-dependent Na channel inhibitors used as local anesthetic, antiarrhythmic, and anticonvulsant drugs. PMID:23486968

Role of action potential configuration and the contribution of Ca2+ and K+ currents to isoprenaline-induced changes in canine ventricular cells

PubMed Central

Szentandrássy, N; Farkas, V; Bárándi, L; Hegyi, B; Ruzsnavszky, F; Horváth, B; Bányász, T; Magyar, J; Márton, I; Nánási, PP

2012-01-01

BACKGROUND AND PURPOSE Although isoprenaline (ISO) is known to activate several ion currents in mammalian myocardium, little is known about the role of action potential morphology in the ISO-induced changes in ion currents. Therefore, the effects of ISO on action potential configuration, L-type Ca2+ current (ICa), slow delayed rectifier K+ current (IKs) and fast delayed rectifier K+ current (IKr) were studied and compared in a frequency-dependent manner using canine isolated ventricular myocytes from various transmural locations. EXPERIMENTAL APPROACH Action potentials were recorded with conventional sharp microelectrodes; ion currents were measured using conventional and action potential voltage clamp techniques. KEY RESULTS In myocytes displaying a spike-and-dome action potential configuration (epicardial and midmyocardial cells), ISO caused reversible shortening of action potentials accompanied by elevation of the plateau. ISO-induced action potential shortening was absent in endocardial cells and in myocytes pretreated with 4-aminopyridine. Application of the IKr blocker E-4031 failed to modify the ISO effect, while action potentials were lengthened by ISO in the presence of the IKs blocker HMR-1556. Both action potential shortening and elevation of the plateau were prevented by pretreatment with the ICa blocker nisoldipine. Action potential voltage clamp experiments revealed a prominent slowly inactivating ICa followed by a rise in IKs, both currents increased with increasing the cycle length. CONCLUSIONS AND IMPLICATIONS The effect of ISO in canine ventricular cells depends critically on action potential configuration, and the ISO-induced activation of IKs– but not IKr– may be responsible for the observed shortening of action potentials. PMID:22563726

Role of action potential configuration and the contribution of C²⁺a and K⁺ currents to isoprenaline-induced changes in canine ventricular cells.

PubMed

Szentandrássy, N; Farkas, V; Bárándi, L; Hegyi, B; Ruzsnavszky, F; Horváth, B; Bányász, T; Magyar, J; Márton, I; Nánási, P P

2012-10-01

Although isoprenaline (ISO) is known to activate several ion currents in mammalian myocardium, little is known about the role of action potential morphology in the ISO-induced changes in ion currents. Therefore, the effects of ISO on action potential configuration, L-type Ca²⁺ current (I(Ca)), slow delayed rectifier K⁺ current (I(Ks)) and fast delayed rectifier K⁺ current (I(Kr)) were studied and compared in a frequency-dependent manner using canine isolated ventricular myocytes from various transmural locations. Action potentials were recorded with conventional sharp microelectrodes; ion currents were measured using conventional and action potential voltage clamp techniques. In myocytes displaying a spike-and-dome action potential configuration (epicardial and midmyocardial cells), ISO caused reversible shortening of action potentials accompanied by elevation of the plateau. ISO-induced action potential shortening was absent in endocardial cells and in myocytes pretreated with 4-aminopyridine. Application of the I(Kr) blocker E-4031 failed to modify the ISO effect, while action potentials were lengthened by ISO in the presence of the I(Ks) blocker HMR-1556. Both action potential shortening and elevation of the plateau were prevented by pretreatment with the I(Ca) blocker nisoldipine. Action potential voltage clamp experiments revealed a prominent slowly inactivating I(Ca) followed by a rise in I(Ks) , both currents increased with increasing the cycle length. The effect of ISO in canine ventricular cells depends critically on action potential configuration, and the ISO-induced activation of I(Ks) - but not I(Kr) - may be responsible for the observed shortening of action potentials. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Electrical properties associated with wide intercellular clefts in rabbit Purkinje fibres.

PubMed Central

Colatsky, T J; Tsien, R W

1979-01-01

1. Rabbit Purkinje fibres were studied using micro-electrode recordings of electrical activity or a two-micro-electrode voltage clamp. Previous morphological work had suggested that these preparations offer structural advantages for the analysis of ionic permeability mechanisms. 2. Viable preparations could be obtained consistently by exposure to a K glutamate Tyrode solution during excision and recovery. In NaCl Tyrode solution, the action potential showed a large overshoot and fully developed plateau, but no pacemaker depolarization at negative potentials. 3. The passive electrical properties were consistent with morphological evidence for the accessibility of cleft membranes within the cell bundle. Electrotonic responses to intracellular current steps showed the behaviour expected for a simple leaky capacitative cable. Capacitative current transients under voltage clamp were changed very little by an eightfold reduction in the external solution conductivity. 4. Slow current changes attributable to K depletion were small compared to those found in other cardiac preparations. The amount of depletion was close to that predicted by a cleft model which assumed free K diffusion in 1 micron clefts. 5. Step depolarizations over the plateau range of potentials evoked a slow inward current which was resistant to tetrodotoxin but blocked by D600. 6. Strong depolarizations to potentials near 0 mV elicited a transient outward current and a slowly activating late outward current. Both components resembled currents found in sheep or calf Purkinje fibres. 7. These experiments support previous interpretations of slow plateau currents in terms of genuine permeability changes. The rabbit Purkinje fibre may allow various ionic channels to be studied with relatively little interference from radial non-uniformities in membrane potential or ion concentration. Images Fig. 7 PMID:469754

A low-voltage sense amplifier with two-stage operational amplifier clamping for flash memory

NASA Astrophysics Data System (ADS)

Guo, Jiarong

2017-04-01

A low-voltage sense amplifier with reference current generator utilizing two-stage operational amplifier clamp structure for flash memory is presented in this paper, capable of operating with minimum supply voltage at 1 V. A new reference current generation circuit composed of a reference cell and a two-stage operational amplifier clamping the drain pole of the reference cell is used to generate the reference current, which avoids the threshold limitation caused by current mirror transistor in the traditional sense amplifier. A novel reference voltage generation circuit using dummy bit-line structure without pull-down current is also adopted, which not only improves the sense window enhancing read precision but also saves power consumption. The sense amplifier was implemented in a flash realized in 90 nm flash technology. Experimental results show the access time is 14.7 ns with power supply of 1.2 V and slow corner at 125 °C. Project supported by the National Natural Science Fundation of China (No. 61376028).

Increased transient Na+ conductance and action potential output in layer 2/3 prefrontal cortex neurons of the fmr1-/y mouse.

PubMed

Routh, Brandy N; Rathour, Rahul K; Baumgardner, Michael E; Kalmbach, Brian E; Johnston, Daniel; Brager, Darrin H

2017-07-01

Layer 2/3 neurons of the prefrontal cortex display higher gain of somatic excitability, responding with a higher number of action potentials for a given stimulus, in fmr1 -/y mice. In fmr1 -/y L2/3 neurons, action potentials are taller, faster and narrower. Outside-out patch clamp recordings revealed that the maximum Na + conductance density is higher in fmr1 -/y L2/3 neurons. Measurements of three biophysically distinct K + currents revealed a depolarizing shift in the activation of a rapidly inactivating (A-type) K + conductance. Realistic neuronal simulations of the biophysical observations recapitulated the elevated action potential and repetitive firing phenotype. Fragile X syndrome is the most common form of inherited mental impairment and autism. The prefrontal cortex is responsible for higher order cognitive processing, and prefrontal dysfunction is believed to underlie many of the cognitive and behavioural phenotypes associated with fragile X syndrome. We recently demonstrated that somatic and dendritic excitability of layer (L) 5 pyramidal neurons in the prefrontal cortex of the fmr1 -/y mouse is significantly altered due to changes in several voltage-gated ion channels. In addition to L5 pyramidal neurons, L2/3 pyramidal neurons play an important role in prefrontal circuitry, integrating inputs from both lower brain regions and the contralateral cortex. Using whole-cell current clamp recording, we found that L2/3 pyramidal neurons in prefrontal cortex of fmr1 -/y mouse fired more action potentials for a given stimulus compared with wild-type neurons. In addition, action potentials in fmr1 -/y neurons were significantly larger, faster and narrower. Voltage clamp of outside-out patches from L2/3 neurons revealed that the transient Na + current was significantly larger in fmr1 -/y neurons. Furthermore, the activation curve of somatic A-type K + current was depolarized. Realistic conductance-based simulations revealed that these biophysical changes in Na + and K + channel function could reliably reproduce the observed increase in action potential firing and altered action potential waveform. These results, in conjunction with our prior findings on L5 neurons, suggest that principal neurons in the circuitry of the medial prefrontal cortex are altered in distinct ways in the fmr1 -/y mouse and may contribute to dysfunctional prefrontal cortex processing in fragile X syndrome. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

LabPatch, an acquisition and analysis program for patch-clamp electrophysiology.

PubMed

Robinson, T; Thomsen, L; Huizinga, J D

2000-05-01

An acquisition and analysis program, "LabPatch," has been developed for use in patch-clamp research. LabPatch controls any patch-clamp amplifier, acquires and records data, runs voltage protocols, plots and analyzes data, and connects to spreadsheet and database programs. Controls within LabPatch are grouped by function on one screen, much like an oscilloscope front panel. The software is mouse driven, so that the user need only point and click. Finally, the ability to copy data to other programs running in Windows 95/98, and the ability to keep track of experiments using a database, make LabPatch extremely versatile. The system requirements include Windows 95/98, at least a 100-MHz processor and 16 MB RAM, a data acquisition card, digital-to-analog converter, and a patch-clamp amplifier. LabPatch is available free of charge at http://www.fhs.mcmaster.ca/huizinga/.

Effect of protons on the mechanical response of rat muscle nociceptive fibers and neurons in vitro.

PubMed

Hotta, Norio; Kubo, Asako; Mizumura, Kazue

2015-03-01

Strong exercise makes muscle acidic, and painful. The stimulus that activates muscle nociceptors in such instance may be protons. Reportedly, however, not many afferents are excited by protons alone. We, therefore, posited that protons sensitize muscular nociceptors to mechanical stimuli. We examined effects of protons on mechanical sensitivity of muscle nociceptors by single-fiber recording from rat muscle-nerve preparations in vitro and by whole cell patch-clamp recording of mechanically activated (MA) currents from cultured rat dorsal root ganglion neurons. We recorded 38 Aδ- and C-fibers. Their response magnitude was increased by both pH 6.2 and pH 6.8; in addition the mechanical threshold was lowered by pH 6.2. Decrease in the threshold by pH6.2 was also observed in MA currents. Presently observed sensitization by protons could be involved in several types of ischemic muscle pain, and may also be involved in cardiovascular and respiratory controls during exercise. Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Current-Induced Transistor Sensorics with Electrogenic Cells

PubMed Central

Fromherz, Peter

2016-01-01

The concepts of transistor recording of electroactive cells are considered, when the response is determined by a current-induced voltage in the electrolyte due to cellular activity. The relationship to traditional transistor recording, with an interface-induced response due to interactions with the open gate oxide, is addressed. For the geometry of a cell-substrate junction, the theory of a planar core-coat conductor is described with a one-compartment approximation. The fast electrical relaxation of the junction and the slow change of ion concentrations are pointed out. On that basis, various recording situations are considered and documented by experiments. For voltage-gated ion channels under voltage clamp, the effects of a changing extracellular ion concentration and the enhancement/depletion of ion conductances in the adherent membrane are addressed. Inhomogeneous ion conductances are crucial for transistor recording of neuronal action potentials. For a propagating action potential, the effects of an axon-substrate junction and the surrounding volume conductor are distinguished. Finally, a receptor-transistor-sensor is described, where the inhomogeneity of a ligand–activated ion conductance is achieved by diffusion of the agonist and inactivation of the conductance. Problems with regard to a development of reliable biosensors are mentioned. PMID:27120627

Existence of multiple receptors in single neurons: responses of single bullfrog olfactory neurons to many cAMP-dependent and independent odorants.

PubMed

Kashiwayanagi, M; Shimano, K; Kurihara, K

1996-11-04

The responses of single bullfrog olfactory neurons to various odorants were measured with the whole-cell patch clamp which offers direct information on cellular events and with the ciliary recording technique to obtain stable quantitative data from many neurons. A large portion of single olfactory neurons (about 64% and 79% in the whole-cell recording and in the ciliary recording, respectively) responded to many odorants with quite diverse molecular structures, including both odorants previously indicated to be cAMP-dependent (increasing) and independent odorants. One odorant elicited a response in many cells; e.g. hedione and citralva elicited the response in 100% and 92% of total neurons examined with the ciliary recording technique. To confirm that a single neuron carries different receptors or transduction pathways, the cross-adaptation technique was applied to single neurons. Application of hedione to a single neuron after desensitization of the current in response to lyral or citralva induced an inward current with a similar magnitude to that applied alone. It was suggested that most single olfactory neurons carry multiple receptors and at least dual transduction pathways.

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.

Fast calcium transients translate the distribution and conduction of neural activity in different regions of a single sensory neuron.

PubMed

Purali, Nuhan

2017-09-01

In the present study, cytosolic calcium concentration changes were recorded in response to various forms of excitations, using the fluorescent calcium indicator dye OG-BAPTA1 together with the current or voltage clamp methods in stretch receptor neurons of crayfish. A single action potential evoked a rise in the resting calcium level in the axon and axonal hillock, whereas an impulse train or a large saturating current injection would be required to evoke an equivalent response in the dendrite region. Under voltage clamp conditions, amplitude differences between axon and dendrite responses vanished completely. The fast activation time and the modulation of the response by extracellular calcium concentration changes indicated that the evoked calcium transients might be mediated by calcium entry into the cytosol through a voltage-gated calcium channel. The decay of the responses was slow and sensitive to extracellular sodium and calcium concentrations as well as exposure to 1-10 mM NiCl 2 and 10-500 µM lanthanum. Thus, a sodium calcium exchanger and a calcium ATPase might be responsible for calcium extrusion from the cytosol. Present results indicate that the calcium indicator OG-BAPTA1 might be an efficient but indirect way of monitoring regional membrane potential differences in a single neuron.

Effects of Imperfect Dynamic Clamp: Computational and Experimental Results

PubMed Central

Bettencourt, Jonathan C.; Lillis, Kyle P.; White, John A.

2008-01-01

In the dynamic clamp technique, a typically nonlinear feedback system delivers electrical current to an excitable cell that represents the actions of “virtual” ion channels (e.g., channels that are gated by local membrane potential or by electrical activity in neighboring biological or virtual neurons). Since the conception of this technique, there have been a number of different implementations of dynamic clamp systems, each with differing levels of flexibility and performance. Embedded hardware-based systems typically offer feedback that is very fast and precisely timed, but these systems are often expensive and sometimes inflexible. PC-based systems, on the other hand, allow the user to write software that defines an arbitrarily complex feedback system, but real-time performance in PC-based systems can be deteriorated by imperfect real-time performance. Here we systematically evaluate the performance requirements for artificial dynamic clamp knock-in of transient sodium and delayed rectifier potassium conductances. Specifically we examine the effects of controller time step duration, differential equation integration method, jitter (variability in time step), and latency (the time lag from reading inputs to updating outputs). Each of these control system flaws is artificially introduced in both simulated and real dynamic clamp experiments. We demonstrate that each of these errors affect dynamic clamp accuracy in a way that depends on the time constants and stiffness of the differential equations being solved. In simulations, time steps above 0.2 ms lead to catastrophic alteration of spike shape, but the frequency-vs.-current relationship is much more robust. Latency (the part of the time step that occurs between measuring membrane potential and injecting re-calculated membrane current) is a crucial factor as well. Experimental data are substantially more sensitive to inaccuracies than simulated data. PMID:18076999

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.

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

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

Planar MEMS bio-chip for recording ion-channel currents in biological cells

NASA Astrophysics Data System (ADS)

Pandey, Santosh; Ferdous, Zannatul; White, Marvin H.

2003-10-01

We describe a planar MEMS silicon structure to record ion-channel currents in biological cells. The conventional method of performing an electrophysiological experiment, 'patch-clamping,' employs a glass micropipette. Despite careful treatments of the micropipette tip, such as fire polishing and surface coating, the latter is a source of thermal noise because of its inherent, tapered, conical structure, which gives rise to a large pipette resistance. This pipette resistance, when coupled with the self-capacitance of the biological cell, limits the available bandwidth and processing of fast transient, ion channel current pulses. In this work, we reduce considerably the pipette resistance with a planar micropipette on a silicon chip to permit the resolution of sub-millisecond, ion-channel pulses. We discuss the design topology of the device, describe the fabrication sequence, and highlight important critical issues. The design of an integrated on-chip CMOS instrumentation amplifier is described, which has a low-noise front-end, input-offset cancellation, correlated double sampling (CDS), and an ultra-high gain in the order of 1012V/A.

High dendritic expression of Ih in the proximity of the axon origin controls the integrative properties of nigral dopamine neurons.

PubMed

Engel, Dominique; Seutin, Vincent

2015-11-15

The hyperpolarization-activated cation current Ih is expressed in dopamine neurons of the substantia nigra, but the subcellular distribution of the current and its role in synaptic integration remain unknown. We used cell-attached patch recordings to determine the localization profile of Ih along the somatodendritic axis of nigral dopamine neurons in slices from young rats. Ih density is higher in axon-bearing dendrites, in a membrane area close to the axon origin, than in the soma and axon-lacking dendrites. Dual current-clamp recordings revealed a similar contribution of Ih to the waveform of single excitatory postsynaptic potentials throughout the somatodendritic domain. The Ih blocker ZD 7288 increased the temporal summation in all dendrites with a comparable effect in axon- and non-axon dendrites. The strategic position of Ih in the proximity of the axon may influence importantly transitions between pacemaker and bursting activities and consequently the downstream release of dopamine. Dendrites of most neurons express voltage-gated ion channels in their membrane. In combination with passive properties, active currents confer to dendrites a high computational potential. The hyperpolarization-activated cation current Ih present in the dendrites of some pyramidal neurons affects their membrane and integration properties, synaptic plasticity and higher functions such as memory. A gradient of increasing h-channel density towards distal dendrites has been found to be responsible for the location independence of excitatory postsynaptic potential (EPSP) waveform and temporal summation in cortical and hippocampal pyramidal cells. However, reports on other cell types revealed that smoother gradients or even linear distributions of Ih can achieve homogeneous temporal summation. Although the existence of a robust, slowly activating Ih current has been repeatedly demonstrated in nigral dopamine neurons, its subcellular distribution and precise role in synaptic integration are unknown. Using cell-attached patch-clamp recordings, we find a higher Ih current density in the axon-bearing dendrite than in the soma or in dendrites without axon in nigral dopamine neurons. Ih is mainly concentrated in the dendritic membrane area surrounding the axon origin and decreases with increasing distances from this site. Single EPSPs and temporal summation are similarly affected by blockade of Ih in axon- and non-axon-bearing dendrites. The presence of Ih close to the axon is pivotal to control the integrative functions and the output signal of dopamine neurons and may consequently influence the downstream coding of movement. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Identification of acid-sensing ion channels in adenoid cystic carcinomas

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

Ye Jinhai; Department of Oral and Maxillofacial Surgery, School of Stomatology, Nanjing Medical University, Research Institute of Stomatology, Nanjing 210029; Gao Jun

2007-04-20

Tissue acidosis is an important feature of tumor. The response of adenoid cystic carcinoma (ACC) cells to acidic solution was studied using whole-cell patch-clamp recording in the current study. An inward, amiloride-sensitive Na{sup +} current was identified in cultured ACC-2 cells while not in normal human salivary gland epithelial cells. Electrophysiological and pharmacological properties of the currents suggest that heteromeric acid-sensing ion channels (ASICs) containing 2a and 3 may be responsible for the proton-induced currents in the majority of ACC-2 cells. Consistent with it, analyses of RT-PCR and Western blotting demonstrated the presences of ASIC2a and 3 in ACC-2 cells.more » Furthermore, we observed the enhanced expression of ASIC2a and 3 in the sample of ACC tissues. These results indicate that the functional expression of ASICs is characteristic feature of ACC cells.« less

Crebanine inhibits voltage-dependent Na+ current in guinea-pig ventricular myocytes.

PubMed

Xiao-Shan, He; Qing, Lin; Yun-Shu, Ma; Ze-Pu, Yu

2014-01-01

To study the effects of crebanine on voltage-gated Na(+) channels in cardiac tissues. Single ventricular myocytes were enzymatically dissociated from adult guinea-pig heart. Voltage-dependent Na(+) current was recorded using the whole cell voltage-clamp technique. Crebanine reversibly inhibited Na(+) current with an IC50 value of 0.283 mmol·L(-1) (95% confidence range: 0.248-0.318 mmol·L(-1)). Crebanine at 0.262 mmol·L(-1) caused a negative shift (about 12 mV) in the voltage-dependence of steady-state inactivation of Na(+) current, and retarded its recovery from inactivation, but did not affect its activation curve. In addition to blocking other voltage-gated ion channels, crebanine blocked Na(+) channels in guinea-pig ventricular myocytes. Crebanine acted as an inactivation stabilizer of Na(+) channels in cardiac tissues. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Functional reconstitution of the voltage-regulated sodium channel purified from electroplax of Electrophorus electricus

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

Rosenberg, R.L.

1985-01-01

The voltage-regulated NA channel is responsible for the depolarization of the excitable cell membrane during the normal action potential. This research has focused on the functional properties of the Na channel, purified from detergent extracts of electroplax membranes of the electric eel, and reconstituted into vesicles of defined phospholipid. These properties were assessed by measuring neurotoxin-modulated ion flux into the reconstituted membrane vesicles and by recording the single-channel currents of the purified channel by the patch-clamp method. The binding of tritiated tetrodotoxin (TTX) was employed as a marker for the purification of the channel. Two high-resolution fractionation steps, based onmore » molecular charge and protein size, were used to obtain a preparation that is 80% homogeneous for a large peptide of 270,000 daltons. Radiotracer /sup 22/Na/sup +/ influx into the vesicles was stimulated by veratridine and by batrachotoxin (BTX) at concentrations of 100 ..mu..M and 5 ..mu..M, respectively. The stimulation by BTX was greater than that by veratridine, and can be as much as 16-fold over control influx levels. The stimulated influx is blocked by TTX with a K/sub i/ of 35 nM, and by local anesthetics in the normal pharmacological range. Large multilamellar vesicles prepared with a freeze-thaw step are suitable for single-channel recording techniques. When excised patches of the reconstituted membranes were voltage-clamped in the absence of activating neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties similar to those from native membranes of nerve and muscle. These results indicate that the protein purified on the basis of TTX binding is a functional Na channel possessing these functional domains: the ion-selective channel, the voltage sensors controlling activation and inactivation, and the sites of action of TTX, alkaloid neurotoxins, and local anesthetics.« less

Role of plasma membrane-associated AKAPs for the regulation of cardiac IK1 current by protein kinase A.

PubMed

Seyler, Claudia; Scherer, Daniel; Köpple, Christoph; Kulzer, Martin; Korkmaz, Sevil; Xynogalos, Panagiotis; Thomas, Dierk; Kaya, Ziya; Scholz, Eberhard; Backs, Johannes; Karle, Christoph; Katus, Hugo A; Zitron, Edgar

2017-05-01

The cardiac I K1 current stabilizes the resting membrane potential of cardiomyocytes. Protein kinase A (PKA) induces an inhibition of I K1 current which strongly promotes focal arrhythmogenesis. The molecular mechanisms underlying this regulation have only partially been elucidated yet. Furthermore, the role of A-kinase anchoring proteins (AKAPs) in this regulation has not been examined to date. The objective of this project was to elucidate the molecular mechanisms underlying the inhibition of I K1 by PKA and to identify novel molecular targets for antiarrhythmic therapy downstream β-adrenoreceptors. Patch clamp and voltage clamp experiments were used to record currents and co-immunoprecipitation, and co-localization experiments were performed to show spatial and functional coupling. Activation of PKA inhibited I K1 current in rat cardiomyocytes. This regulation was markedly attenuated by disrupting PKA-binding to AKAPs with the peptide inhibitor AKAP-IS. We observed functional and spatial coupling of the plasma membrane-associated AKAP15 and AKAP79 to Kir2.1 and Kir2.2 channel subunits, but not to Kir2.3 channels. In contrast, AKAPyotiao had no functional effect on the PKA regulation of Kir channels. AKAP15 and AKAP79 co-immunoprecipitated with and co-localized to Kir2.1 and Kir2.2 channel subunits in ventricular cardiomyocytes. In this study, we provide evidence for coupling of cardiac Kir2.1 and Kir2.2 subunits with the plasma membrane-bound AKAPs 15 and 79. Cardiac membrane-associated AKAPs are a functionally essential part of the regulatory cascade determining I K1 current function and may be novel molecular targets for antiarrhythmic therapy downstream from β-adrenoreceptors.

Effect of cholesterol depletion on the pore dilation of TRPV1.

PubMed

Jansson, Erik T; Trkulja, Carolina L; Ahemaiti, Aikeremu; Millingen, Maria; Jeffries, Gavin Dm; Jardemark, Kent; Orwar, Owe

2013-01-02

The TRPV1 ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a means of alleviating pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate the effects of cholesterol depletion of the cell on ion-permeability of the TRPV1 ion channel. The ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies on a Chinese hamster ovary (CHO) cell line expressing this ion channel. Prolonged capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole extracellular cation, generated a biphasic current which included an initial outward current followed by an inward current. Similarly, prolonged proton-activation (pH 5.5) of TRPV1 under hypocalcemic conditions also generated a biphasic current including a fast initial current peak followed by a larger second one. Patch-clamp recordings of reversal potentials of TRPV1 revealed an increase of the ion-permeability for NMDG during prolonged activation of this ion channel under hypocalcemic conditions. Our findings show that cholesterol depletion inhibited both the second current, and the increase in ion-permeability of the TRPV1 channel, resulting from sustained agonist-activation with capsaicin and protons (pH 5.5). These results were confirmed with YO-PRO uptake rate studies using laser scanning confocal microscopy, where cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Hence, these results propose a novel mechanism by which cellular cholesterol depletion modulates the function of TRPV1, which may constitute a novel approach for treatment of neurogenic pain.

Basally activated nonselective cation currents regulate the resting membrane potential in human and monkey colonic smooth muscle

PubMed Central

Dwyer, Laura; Rhee, Poong-Lyul; Lowe, Vanessa; Zheng, Haifeng; Peri, Lauren; Ro, Seungil; Sanders, Kenton M.

2011-01-01

Resting membrane potential (RMP) plays an important role in determining the basal excitability of gastrointestinal smooth muscle. The RMP in colonic muscles is significantly less negative than the equilibrium potential of K+, suggesting that it is regulated not only by K+ conductances but by inward conductances such as Na+ and/or Ca2+. We investigated the contribution of nonselective cation channels (NSCC) to the RMP in human and monkey colonic smooth muscle cells (SMC) using voltage- and current-clamp techniques. Qualitative reverse transcriptase-polymerase chain reaction was performed to examine potential molecular candidates for these channels among the transient receptor potential (TRP) channel superfamily. Spontaneous transient inward currents and holding currents were recorded in human and monkey SMC. Replacement of extracellular Na+ with equimolar tetraethylammonium or Ca2+ with Mn2+ inhibited basally activated nonselective cation currents. Trivalent cations inhibited these channels. Under current clamp, replacement of extracellular Na+ with N-methyl-d-glucamine or addition of trivalent cations caused hyperpolarization. Three unitary conductances of NSCC were observed in human and monkey colonic SMC. Molecular candidates for basally active NSCC were TRPC1, C3, C4, C7, M2, M4, M6, M7, V1, and V2 in human and monkey SMC. Comparison of the biophysical properties of these TRP channels with basally active NSCC (bINSCC) suggests that TRPM4 and specific TRPC heteromultimer combinations may underlie the three single-channel conductances of bINSCC. In conclusion, these findings suggest that basally activated NSCC contribute to the RMP in human and monkey colonic SMC and therefore may play an important role in determining basal excitability of colonic smooth muscle. PMID:21566016

MATLAB-based automated patch-clamp system for awake behaving mice

PubMed Central

Siegel, Jennifer J.; Taylor, William; Chitwood, Raymond A.; Johnston, Daniel

2015-01-01

Automation has been an important part of biomedical research for decades, and the use of automated and robotic systems is now standard for such tasks as DNA sequencing, microfluidics, and high-throughput screening. Recently, Kodandaramaiah and colleagues (Nat Methods 9: 585–587, 2012) demonstrated, using anesthetized animals, the feasibility of automating blind patch-clamp recordings in vivo. Blind patch is a good target for automation because it is a complex yet highly stereotyped process that revolves around analysis of a single signal (electrode impedance) and movement along a single axis. Here, we introduce an automated system for blind patch-clamp recordings from awake, head-fixed mice running on a wheel. In its design, we were guided by 3 requirements: easy-to-use and easy-to-modify software; seamless integration of behavioral equipment; and efficient use of time. The resulting system employs equipment that is standard for patch recording rigs, moderately priced, or simple to make. It is written entirely in MATLAB, a programming environment that has an enormous user base in the neuroscience community and many available resources for analysis and instrument control. Using this system, we obtained 19 whole cell patch recordings from neurons in the prefrontal cortex of awake mice, aged 8–9 wk. Successful recordings had series resistances that averaged 52 ± 4 MΩ and required 5.7 ± 0.6 attempts to obtain. These numbers are comparable with those of experienced electrophysiologists working manually, and this system, written in a simple and familiar language, will be useful to many cellular electrophysiologists who wish to study awake behaving mice. PMID:26084901

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.

Auxiliary quasi-resonant dc tank electrical power converter

DOEpatents

Peng, Fang Z.

2006-10-24

An auxiliary quasi-resonant dc tank (AQRDCT) power converter with fast current charging, voltage balancing (or charging), and voltage clamping circuits is provided for achieving soft-switched power conversion. The present invention is an improvement of the invention taught in U.S. Pat. No. 6,111,770, herein incorporated by reference. The present invention provides faster current charging to the resonant inductor, thus minimizing delay time of the pulse width modulation (PWM) due to the soft-switching process. The new AQRDCT converter includes three tank capacitors or power supplies to achieve the faster current charging and minimize the soft-switching time delay. The new AQRDCT converter further includes a voltage balancing circuit to charge and discharge the three tank capacitors so that additional isolated power supplies from the utility line are not needed. A voltage clamping circuit is also included for clamping voltage surge due to the reverse recovery of diodes.

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

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

Membrane potential dynamics of axons in cultured hippocampal neurons probed by second-harmonic-generation imaging

NASA Astrophysics Data System (ADS)

Nuriya, Mutsuo; Yasui, Masato

2010-03-01

The electrical properties of axons critically influence the nature of communication between neurons. However, due to their small size, direct measurement of membrane potential dynamics in intact and complex mammalian axons has been a challenge. Furthermore, quantitative optical measurements of axonal membrane potential dynamics have not been available. To characterize the basic principles of somatic voltage signal propagation in intact axonal arbors, second-harmonic-generation (SHG) imaging is applied to cultured mouse hippocampal neurons. When FM4-64 is applied extracellularly to dissociated neurons, whole axonal arbors are visualized by SHG imaging. Upon action potential generation by somatic current injection, nonattenuating action potentials are recorded in intact axonal arbors. Interestingly, however, both current- and voltage-clamp recordings suggest that nonregenerative subthreshold somatic voltage changes at the soma are poorly conveyed to these axonal sites. These results reveal the nature of membrane potential dynamics of cultured hippocampal neurons, and further show the possibility of SHG imaging in physiological investigations of axons.

Command-line cellular electrophysiology for conventional and real-time closed-loop experiments.

PubMed

Linaro, Daniele; Couto, João; Giugliano, Michele

2014-06-15

Current software tools for electrophysiological experiments are limited in flexibility and rarely offer adequate support for advanced techniques such as dynamic clamp and hybrid experiments, which are therefore limited to laboratories with a significant expertise in neuroinformatics. We have developed lcg, a software suite based on a command-line interface (CLI) that allows performing both standard and advanced electrophysiological experiments. Stimulation protocols for classical voltage and current clamp experiments are defined by a concise and flexible meta description that allows representing complex waveforms as a piece-wise parametric decomposition of elementary sub-waveforms, abstracting the stimulation hardware. To perform complex experiments lcg provides a set of elementary building blocks that can be interconnected to yield a large variety of experimental paradigms. We present various cellular electrophysiological experiments in which lcg has been employed, ranging from the automated application of current clamp protocols for characterizing basic electrophysiological properties of neurons, to dynamic clamp, response clamp, and hybrid experiments. We finally show how the scripting capabilities behind a CLI are suited for integrating experimental trials into complex workflows, where actual experiment, online data analysis and computational modeling seamlessly integrate. We compare lcg with two open source toolboxes, RTXI and RELACS. We believe that lcg will greatly contribute to the standardization and reproducibility of both simple and complex experiments. Additionally, on the long run the increased efficiency due to a CLI will prove a great benefit for the experimental community. Copyright © 2014 Elsevier B.V. All rights reserved.

Sodium efflux from voltage clamped squid giant axons.

PubMed Central

Landowne, D

1977-01-01

1. The efflux of radioactive sodium was measured from squid axons during simultaneous voltage clamp experiments such that it was possible to determine the efflux of sodium associated with a measured voltage clamp current. 2. The extra efflux of sodium associated with voltage clamp pulses increased linearly with the magnitude of the depolarization above 40 mV. A 100 mV pulse of sufficient duration to produce all of the sodium current increased the rate constant of efflux by about 10(-6). 3. Application of 100 nM tetrodotoxin eliminated the sodium current and the extra efflux of radioactive sodium. 4. Cooling the axon increased the extra efflux/voltage clamp pulse slightly with a Q10 of 1/1-1. On the same axons cooling increased the integral of the sodium current with a Q10 of 1/1-4. 5. Replacing external sodium with Tris, dextrose or Mg-mannitol reduced the extra efflux of sodium by about 50%. The inward sodium current was replaced with an outward current as expected. 6. Replacing external sodium with lithium also reduced the extra efflux by about 50% but the currents seen in lithium were slightly larger than those in sodium. 7. The effect of replacing external sodium was not voltage dependent. Cooling reduced the effect so that there was less reduction of efflux on switching to Tris ASW in the cold than in the warm. 8. The extra efflux of sodium into sodium-free ASW is approximately the same as the integral of the sodium current. Adding external sodium produces a deviation from the independence principle such that there is more exchange of sodium than predicted. Such a deviation from prediction was noted by Hodgkin & Huxley (1952c). 9. Using the equations of Hodgkin & Huxley (1952c) modified to include the deviation from independence reported in this paper and its temperature dependence, one can predict the temperature dependence of the sodium efflux associated with action potentials and obtain much better agreement than is possibly without these phenomena. 10. This deviation from independence in the sodium fluxes is the type expected from some kind of mixing and binding of sodium within the membrane phase. PMID:856999

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.

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.

Human Nav1.8: enhanced persistent and ramp currents contribute to distinct firing properties of human DRG neurons

PubMed Central

Han, Chongyang; Estacion, Mark; Huang, Jianying; Vasylyev, Dymtro; Zhao, Peng; Dib-Hajj, Sulayman D.

2015-01-01

Although species-specific differences in ion channel properties are well-documented, little has been known about the properties of the human Nav1.8 channel, an important contributor to pain signaling. Here we show, using techniques that include voltage clamp, current clamp, and dynamic clamp in dorsal root ganglion (DRG) neurons, that human Nav1.8 channels display slower inactivation kinetics and produce larger persistent current and ramp current than previously reported in other species. DRG neurons expressing human Nav1.8 channels unexpectedly produce significantly longer-lasting action potentials, including action potentials with half-widths in some cells >10 ms, and increased firing frequency compared with the narrower and usually single action potentials generated by DRG neurons expressing rat Nav1.8 channels. We also show that native human DRG neurons recapitulate these properties of Nav1.8 current and the long-lasting action potentials. Together, our results demonstrate strikingly distinct properties of human Nav1.8, which contribute to the firing properties of human DRG neurons. PMID:25787950

Human Na(v)1.8: enhanced persistent and ramp currents contribute to distinct firing properties of human DRG neurons.

PubMed

Han, Chongyang; Estacion, Mark; Huang, Jianying; Vasylyev, Dymtro; Zhao, Peng; Dib-Hajj, Sulayman D; Waxman, Stephen G

2015-05-01

Although species-specific differences in ion channel properties are well-documented, little has been known about the properties of the human Nav1.8 channel, an important contributor to pain signaling. Here we show, using techniques that include voltage clamp, current clamp, and dynamic clamp in dorsal root ganglion (DRG) neurons, that human Na(v)1.8 channels display slower inactivation kinetics and produce larger persistent current and ramp current than previously reported in other species. DRG neurons expressing human Na(v)1.8 channels unexpectedly produce significantly longer-lasting action potentials, including action potentials with half-widths in some cells >10 ms, and increased firing frequency compared with the narrower and usually single action potentials generated by DRG neurons expressing rat Na(v)1.8 channels. We also show that native human DRG neurons recapitulate these properties of Na(v)1.8 current and the long-lasting action potentials. Together, our results demonstrate strikingly distinct properties of human Na(v)1.8, which contribute to the firing properties of human DRG neurons.

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.

Hyperpolarizing muscarinic responses of freshly dissociated rat hippocampal CA1 neurones.

PubMed Central

Wakamori, M; Hidaka, H; Akaike, N

1993-01-01

1. Intracellular mechanisms of the muscarinic acetylcholine (ACh) response were investigated in pyramidal neurones freshly dissociated from the rat hippocampal CA1 region. Current recordings were made in the whole-cell mode using the nystatin 'perforated'-patch technique, by which the muscarinic ACh response can be continuously recorded without so-called 'run-down' phenomenon. The amount of intracellular free Ca2+ ([Ca2+]i) was fluorometrically measured using fura-2. 2. In current clamp conditions, ACh induced a transient hyperpolarization accompanied by a decrease in membrane input resistance. 3. Under voltage clamp conditions at a holding potential (Vh) of -40 mV, ACh induced two types of muscarinic currents observed either alone or together: a transient outward current and a slowly activating sustained inward current. 4. The ACh-induced transient outward current reversed the direction at K+ equilibrium potential (EK), and the reversal potential (EACh) shifted 56.7 mV for a tenfold change of extracellular K+ concentration ([K+]o). 5. The ACh-induced transient outward current increased in a sigmoidal fashion with increase in ACh concentration, where the half-maximal concentration (EC50) and the Hill coefficient (n) were 8 x 10(-7) M and 1.9, respectively. Both muscarine and carbamylcholine mimicked the ACh response, but neither McN-A-343 (M1 agonist) nor oxotremorine (cardiac M2 agonist) induced any current. 6. Muscarinic antagonists reversibly blocked the ACh response in a concentration-dependent manner. The inhibitory potency was in the order of atropine > pirenzepine > AF-DX-116. 7. The ACh-induced transient outward current was never recorded when [Ca2+]i was chelated by the acetoxymethyl ester form of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA AM). On the other hand, in Ca(2+)-free external solution containing 2 mM EGTA and 10 mM Mg2+, the ACh response was elicited by the first application and successive ACh applications did not induce any response. Fura-2 imaging showed that [Ca2+]i was increased when ACh was added to the external medium with or without Ca2+, though in Ca(2+)-free medium only the first application of ACh increased the [Ca2+]i. 8. The ACh response was not affected by pretreatment with pertussis toxin (PTX) but the inhibitory effect of ACh on the high-threshold Ca2+ channel was abolished completely. 9. Pretreatment with Li+ enhanced the amplitude of the transient outward current and the increase in [Ca2+]i induced by ACh. 10. The calmodulin antagonists W-7, chlorpromazine and trifluoperazine reversibly inhibited the ACh response in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7504109

Activity of the anticonvulsant lacosamide in experimental and human epilepsy via selective effects on slow Na+ channel inactivation.

PubMed

Holtkamp, Dominik; Opitz, Thoralf; Niespodziany, Isabelle; Wolff, Christian; Beck, Heinz

2017-01-01

In human epilepsy, pharmacoresistance to antiepileptic drug therapy is a major problem affecting ~30% of patients with epilepsy. Many classical antiepileptic drugs target voltage-gated sodium channels, and their potent activity in inhibiting high-frequency firing has been attributed to their strong use-dependent blocking action. In chronic epilepsy, a loss of use-dependent block has emerged as a potential cellular mechanism of pharmacoresistance for anticonvulsants acting on voltage-gated sodium channels. The anticonvulsant drug lacosamide (LCM) also targets sodium channels, but has been shown to preferentially affect sodium channel slow inactivation processes, in contrast to most other anticonvulsants. We used whole-cell voltage clamp recordings in acutely isolated cells to investigate the effects of LCM on transient Na + currents. Furthermore, we used whole-cell current clamp recordings to assess effects on repetitive action potential firing in hippocampal slices. We show here that LCM exerts its effects primarily via shifting the slow inactivation voltage dependence to more hyperpolarized potentials in hippocampal dentate granule cells from control and epileptic rats, and from patients with epilepsy. It is important to note that this activity of LCM was maintained in chronic experimental and human epilepsy. Furthermore, we demonstrate that the efficacy of LCM in inhibiting high-frequency firing is undiminished in chronic experimental and human epilepsy. Taken together, these results show that LCM exhibits maintained efficacy in chronic epilepsy, in contrast to conventional use-dependent sodium channel blockers such as carbamazepine. They also establish that targeting slow inactivation may be a promising strategy for overcoming target mechanisms of pharmacoresistance. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

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

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.

Inhibition of spontaneous activity of rabbit atrioventricular node cells by KB-R7943 and inhibitors of sarcoplasmic reticulum Ca2+ ATPase

PubMed Central

Cheng, Hongwei; Smith, Godfrey L.; Hancox, Jules C.; Orchard, Clive H.

2011-01-01

The atrioventricular node (AVN) can act as a subsidiary cardiac pacemaker if the sinoatrial node fails. In this study, we investigated the effects of the Na–Ca exchange (NCX) inhibitor KB-R7943, and inhibition of the sarcoplasmic reticulum calcium ATPase (SERCA), using thapsigargin or cyclopiazonic acid (CPA), on spontaneous action potentials (APs) and [Ca2+]i transients from cells isolated from the rabbit AVN. Spontaneous [Ca2+]i transients were monitored from undialysed AVN cells at 37 °C using Fluo-4. In separate experiments, spontaneous APs and ionic currents were recorded using the whole-cell patch clamp technique. Rapid application of 5 μM KB-R7943 slowed or stopped spontaneous APs and [Ca2+]i transients. However, in voltage clamp experiments in addition to blocking NCX current (INCX) KB-R7943 partially inhibited L-type calcium current (ICa,L). Rapid reduction of external [Na+] also abolished spontaneous activity. Inhibition of SERCA (using 2.5 μM thapsigargin or 30 μM CPA) also slowed or stopped spontaneous APs and [Ca2+]i transients. Our findings are consistent with the hypothesis that sarcoplasmic reticulum (SR) Ca2+ release influences spontaneous activity in AVN cells, and that this occurs via [Ca2+]i-activated INCX; however, the inhibitory action of KB-R7943 on ICa,L means that care is required in the interpretation of data obtained using this compound. PMID:21163524

Control of Phasic Firing by a Background Leak Current in Avian Forebrain Auditory Neurons

PubMed Central

Dagostin, André A.; Lovell, Peter V.; Hilscher, Markus M.; Mello, Claudio V.; Leão, Ricardo M.

2015-01-01

Central neurons express a variety of neuronal types and ion channels that promote firing heterogeneity among their distinct neuronal populations. Action potential (AP) phasic firing, produced by low-threshold voltage-activated potassium currents (VAKCs), is commonly observed in mammalian brainstem neurons involved in the processing of temporal properties of the acoustic information. The avian caudomedial nidopallium (NCM) is an auditory area analogous to portions of the mammalian auditory cortex that is involved in the perceptual discrimination and memorization of birdsong and shows complex responses to auditory stimuli We performed in vitro whole-cell patch-clamp recordings in brain slices from adult zebra finches (Taeniopygia guttata) and observed that half of NCM neurons fire APs phasically in response to membrane depolarizations, while the rest fire transiently or tonically. Phasic neurons fired APs faster and with more temporal precision than tonic and transient neurons. These neurons had similar membrane resting potentials, but phasic neurons had lower membrane input resistance and time constant. Surprisingly phasic neurons did not express low-threshold VAKCs, which curtailed firing in phasic mammalian brainstem neurons, having similar VAKCs to other NCM neurons. The phasic firing was determined not by VAKCs, but by the potassium background leak conductances, which was more prominently expressed in phasic neurons, a result corroborated by pharmacological, dynamic-clamp, and modeling experiments. These results reveal a new role for leak currents in generating firing diversity in central neurons. PMID:26696830

FPGA in-the-loop simulations of cardiac excitation model under voltage clamp conditions

NASA Astrophysics Data System (ADS)

Othman, Norliza; Adon, Nur Atiqah; Mahmud, Farhanahani

2017-01-01

Voltage clamp technique allows the detection of single channel currents in biological membranes in identifying variety of electrophysiological problems in the cellular level. In this paper, a simulation study of the voltage clamp technique has been presented to analyse current-voltage (I-V) characteristics of ion currents based on Luo-Rudy Phase-I (LR-I) cardiac model by using a Field Programmable Gate Array (FPGA). Nowadays, cardiac models are becoming increasingly complex which can cause a vast amount of time to run the simulation. Thus, a real-time hardware implementation using FPGA could be one of the best solutions for high-performance real-time systems as it provides high configurability and performance, and able to executes in parallel mode operation. For shorter time development while retaining high confidence results, FPGA-based rapid prototyping through HDL Coder from MATLAB software has been used to construct the algorithm for the simulation system. Basically, the HDL Coder is capable to convert the designed MATLAB Simulink blocks into hardware description language (HDL) for the FPGA implementation. As a result, the voltage-clamp fixed-point design of LR-I model has been successfully conducted in MATLAB Simulink and the simulation of the I-V characteristics of the ionic currents has been verified on Xilinx FPGA Virtex-6 XC6VLX240T development board through an FPGA-in-the-loop (FIL) simulation.

Theoretical investigation of gain-clamped semiconductor optical amplifiers using the amplified spontaneous emission compensating effect

NASA Astrophysics Data System (ADS)

Jia, Xin-Hong

2006-12-01

The theoretical model on gain-clamped semiconductor optical amplifiers (GC-SOAs) based on compensating light has been constructed. Using this model, the effects of insertion position and peak reflectivity of the fiber Bragg grating (FBG) on the gain clamping and noise figure (NF) characteristics of GC-SOA are analyzed. The results show that the effect of the FBG insertion position on gain clamping is slight, but the lower NF can be obtained for input FBG-type GC-SOA; when the FBG peak wavelength is designed to close the signal wavelength, the gain clamping and NF characteristics that can be reached are better. Further study shows that, with the increased peak reflectivity of the FBG, the critical input power is broadened and the gain tends to be varied slowly; the larger bias current is helpful to raise gain and decrease the noise figure but is harmful to a gain flatness characteristic.

Plasma Chamber Restraints in Ignitor and Relevant Disruption Analysis

NASA Astrophysics Data System (ADS)

Gasparotto, M.; Cucchiaro, A.; Capriccioli, A.; Celentano, G.; Rita, C.; Roccella, M.; Macco, B.; Micheli, I.; Ferrari, G.; Orlandi, S.; Coppi, B.

2000-10-01

The plasmas chamber (PC) of Ignitor is made of 12 D-shaped toroidal sectors of Inconel 625 welded together by automatic remote equipment. The thickness of the inboard wall is 17 mm while the middle and outboard walls are 26 mm thick. The PC is supported through the ports by the C-Clamp structure of the toroidal magnet. The main function of the PC supports is to resist the vertical and radial electromagnetic loads and to allow for free movement under thermal loads while providing electrical insulation from the C-Clamps and cryostat. The largest estimated loads are due to a Vertical Displacement Event (VDE) disruption that is followed by a thermal quench and then by the current quench. The vertical supports involve a connection of each radial port to the C-Clamp structure by a link system that withstands the calculated loads. The radial supports resist, with high stiffness, the centripetal and centrifugal forces. The end flange of each radial port is connected to the C-Clamp structure by a clamping sleeve device. The clamping sleeves are hydraulically operated to provide locking during discharge. The clamping sleeves of the radial support system have been validated by an appropriate series of tests.

Light-evoked currents in retinal ganglion cells from dystrophic RCS rats.

PubMed

Liu, Kang; Wang, Yi; Yin, Zhengqin; Weng, Chuanhuang

2013-01-01

To study the electrophysiological properties of the light-evoked currents in ganglion cells in situations of retinal degeneration. We investigated light-evoked currents in ganglion cells by performing whole-cell patch-clamp recordings from ganglion cells using a retina-stretched preparation from Royal College of Surgeons (RCS) rats, a model of retinal degeneration and congenic controls at different ages. Pharmacological inhibitors of the AMPA receptor (NBQX), GABA receptor (BMI), and sodium channels (TTX) were used to identify the components of the light-evoked currents in ON, OFF and ON-OFF retinal ganglion cells. We found that the light-evoked currents in ganglion cells from control rats were inhibited by NBQX, BMI and TTX, suggesting that AMPA receptors, GABA receptors and sodium channels contribute to these currents in ganglion cells. However, only AMPA receptor-mediated currents were recorded in RCS rats. Light-evoked inward currents were absent in the majority of ganglion cells from RCS rats, particularly at the later stages of retinal degeneration. At earlier stages of retinal degeneration, we found that both the timing and amplitude of light-evoked currents are significantly different in ganglion cells from RCS and control rats. Our study furthers the understanding of the electrophysiological characteristics of retinal ganglion cells during retinal degeneration, and provides insight into the optimal timing for the treatment of retinal degeneration. Copyright © 2013 S. Karger AG, Basel.

Sustained and transient calcium currents in horizontal cells of the white bass retina.

PubMed

Sullivan, J M; Lasater, E M

1992-01-01

Calcium currents were recorded from cultured horizontal cells (HCs) isolated from adult white bass retinas, using the whole-cell patch-clamp technique. Ca2+ currents were enhanced using 10 mM extracellular Ca2+, while Na+ and K+ currents were pharmacologically suppressed. Two components of the Ca2+ current, one transient, the other sustained, were found. The large transient component of the Ca2+ current, which has not been seen before in HCs, is similar, but not identical, to the T-type Ca2+ current described previously in a variety of preparations. The sustained component of the Ca2+ current is similar, but not identical, to the L-type current described in other preparations. FTX, a factor isolated from the venom of the funnel-web spider, Agelenopsis aperta, preferentially and irreversibly blocks the sustained component of the Ca2+ current at very dilute concentrations. The sustained component of the Ca2+ current inactivates slowly, over the course of 15-60 s, in some HCs. This inactivation of the sustained Ca2+ current, when present, is primarily voltage dependent rather than Ca2+ dependent.

Sustained and transient calcium currents in horizontal cells of the white bass retina

PubMed Central

1992-01-01

Calcium currents were recorded from cultured horizontal cells (HCs) isolated from adult white bass retinas, using the whole-cell patch- clamp technique. Ca2+ currents were enhanced using 10 mM extracellular Ca2+, while Na+ and K+ currents were pharmacologically suppressed. Two components of the Ca2+ current, one transient, the other sustained, were found. The large transient component of the Ca2+ current, which has not been seen before in HCs, is similar, but not identical, to the T-type Ca2+ current described previously in a variety of preparations. The sustained component of the Ca2+ current is similar, but not identical, to the L-type current described in other preparations. FTX, a factor isolated from the venom of the funnel-web spider, Agelenopsis aperta, preferentially and irreversibly blocks the sustained component of the Ca2+ current at very dilute concentrations. The sustained component of the Ca2+ current inactivates slowly, over the course of 15- 60 s, in some HCs. This inactivation of the sustained Ca2+ current, when present, is primarily voltage dependent rather than Ca2+ dependent. PMID:1371309

[Comparison of validity and safety between holmium: YAG laser and traditional surgery in partial nephrectomy].

PubMed

Bi, Sheng; Xia, Ming

2015-08-11

To compare the validity and safety between holmium: YAG laser and traditional surgery in partial nephrectomy. A total of 28 patients were divided into two groups (holmium: YAG laser group without renal artery clamping and traditional surgery group with renal artery clamping). The intraoperative blood loss, total operative time, renal artery clamping time, postoperative hospital stay, separated renal function, postoperative complications and depth of tissue injury were recorded. The intraoperative blood loss, total operative time, renal artery clamping time, postoperative hospital stay, separated renal function, postoperative complications and depth of tissue injury were 80 ml, 77 min, 0 min, 7.4 days, 35 ml/min, 0, 0.9 cm, respectively, in holmium: YAG laser group. And in traditional surgery group were 69 ml, 111 min, 25.5 min, 7.3 days, 34 ml/min, 0, 2.0 cm, respectively. The differences of total operative time, renal artery clamping time and depth of tissue injury between two groups were statistically significant. The others were not statistically significant. Holmium: YAG laser is effective and safe in partial nephrectomy. It can decrease the total operative time, minimize the warm ischemia time and enlarge the extent of surgical excision.

Timing and efficacy of Ca2+ channel activation in hippocampal mossy fiber boutons.

PubMed

Bischofberger, Josef; Geiger, Jörg R P; Jonas, Peter

2002-12-15

The presynaptic Ca2+ signal is a key determinant of transmitter release at chemical synapses. In cortical synaptic terminals, however, little is known about the kinetic properties of the presynaptic Ca2+ channels. To investigate the timing and magnitude of the presynaptic Ca2+ inflow, we performed whole-cell patch-clamp recordings from mossy fiber boutons (MFBs) in rat hippocampus. MFBs showed large high-voltage-activated Ca(2+) currents, with a maximal amplitude of approximately 100 pA at a membrane potential of 0 mV. Both activation and deactivation were fast, with time constants in the submillisecond range at a temperature of approximately 23 degrees C. An MFB action potential (AP) applied as a voltage-clamp command evoked a transient Ca2+ current with an average amplitude of approximately 170 pA and a half-duration of 580 microsec. A prepulse to +40 mV had only minimal effects on the AP-evoked Ca2+ current, indicating that presynaptic APs open the voltage-gated Ca2+ channels very effectively. On the basis of the experimental data, we developed a kinetic model with four closed states and one open state, linked by voltage-dependent rate constants. Simulations of the Ca2+ current could reproduce the experimental data, including the large amplitude and rapid time course of the current evoked by MFB APs. Furthermore, the simulations indicate that the shape of the presynaptic AP and the gating kinetics of the Ca2+ channels are tuned to produce a maximal Ca2+ influx during a minimal period of time. The precise timing and high efficacy of Ca2+ channel activation at this cortical glutamatergic synapse may be important for synchronous transmitter release and temporal information processing.

Selective activation of heteromeric SK channels contributes to action potential repolarization in mouse atrial myocytes.

PubMed

Hancock, Jane M; Weatherall, Kate L; Choisy, Stéphanie C; James, Andrew F; Hancox, Jules C; Marrion, Neil V

2015-05-01

Activation of small conductance calcium-activated potassium (SK) channels is proposed to contribute to repolarization of the action potential in atrial myocytes. This role is controversial, as these cardiac SK channels appear to exhibit an uncharacteristic pharmacology. The objectives of this study were to resolve whether activation of SK channels contributes to atrial action potential repolarization and to determine the likely subunit composition of the channel. The effect of 2 SK channel inhibitors was assessed on outward current evoked in voltage clamp and on action potential duration in perforated patch and whole-cell current clamp recording from acutely isolated mouse atrial myocytes. The presence of SK channel subunits was assessed using immunocytochemistry. A significant component of outward current was reduced by the SK channel blockers apamin and UCL1684. Block by apamin displayed a sensitivity indicating that this current was carried by homomeric SK2 channels. Action potential duration was significantly prolonged by UCL1684, but not by apamin. This effect was accompanied by an increase in beat-to-beat variability and action potential triangulation. This pharmacology was matched by that of expressed heteromeric SK2-SK3 channels in HEK293 cells. Immunocytochemistry showed that atrial myocytes express both SK2 and SK3 channels with an overlapping expression pattern. Only proposed heteromeric SK2-SK3 channels are physiologically activated to contribute to action potential repolarization, which is indicated by the difference in pharmacology of evoked outward current and prolongation of atrial action potential duration. The effect of blocking this channel on the action potential suggests that SK channel inhibition during cardiac function has the potential to be proarrhythmic. Copyright © 2015 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Action potential bursts in central snail neurons elicited by paeonol: roles of ionic currents

PubMed Central

Chen, Yi-hung; Lin, Pei-lin; Hsu, Hui-yu; Wu, Ya-ting; Yang, Han-yin; Lu, Dah-yuu; Huang, Shiang-suo; Hsieh, Ching-liang; Lin, Jaung-geng

2010-01-01

Aim: To investigate the effects of 2′-hydroxy-4′-methoxyacetophenone (paeonol) on the electrophysiological behavior of a central neuron (right parietal 4; RP4) of the giant African snail (Achatina fulica Ferussac). Methods: Intracellular recordings and the two-electrode voltage clamp method were used to study the effects of paeonol on the RP4 neuron. Results: The RP4 neuron generated spontaneous action potentials. Bath application of paeonol at a concentration of ≥500 μmol/L reversibly elicited action potential bursts in a concentration-dependent manner. Immersing the neurons in Co2+-substituted Ca2+-free solution did not block paeonol-elicited bursting. Pretreatment with the protein kinase A (PKA) inhibitor KT-5720 or the protein kinase C (PKC) inhibitor Ro 31-8220 did not affect the action potential bursts. Voltage-clamp studies revealed that paeonol at a concentration of 500 μmol/L had no remarkable effects on the total inward currents, whereas paeonol decreased the delayed rectifying K+ current (IKD) and the fast-inactivating K+ current (IA). Application of 4-aminopyridine (4-AP 5 mmol/L), an inhibitor of IA, or charybdotoxin 250 nmol/L, an inhibitor of the Ca2+-activated K+ current (IK(Ca)), failed to elicit action potential bursts, whereas tetraethylammonium chloride (TEA 50 mmol/L), an IKD blocker, successfully elicited action potential bursts. At a lower concentration of 5 mmol/L, TEA facilitated the induction of action potential bursts elicited by paeonol. Conclusion: Paeonol elicited a bursting firing pattern of action potentials in the RP4 neuron and this activity relates closely to the inhibitory effects of paeonol on the IKD. PMID:21042287

Inwardly Rectifying K+ Currents in Cultured Oligodendrocytes from Rat Optic Nerve are Insensitive to pH.

PubMed

Pérez-Samartín, Alberto; Garay, Edith; Moctezuma, Juan Pablo H; Cisneros-Mejorado, Abraham; Sánchez-Gómez, María Victoria; Martel-Gallegos, Guadalupe; Robles-Martínez, Leticia; Canedo-Antelo, Manuel; Matute, Carlos; Arellano, Rogelio O

2017-09-01

Inwardly rectifying K + (Kir) channel expression signals at an advanced stage of maturation during oligodendroglial differentiation. Knocking down their expression halts the generation of myelin and produces severe abnormalities in the central nervous system. Kir4.1 is the main subunit involved in the tetrameric structure of Kir channels in glial cells; however, the precise composition of Kir channels expressed in oligodendrocytes (OLs) remains partially unknown, as participation of other subunits has been proposed. Kir channels are sensitive to H + ; thus, intracellular acidification produces Kir current inhibition. Since Kir subunits have differential sensitivity to H + , we studied the effect of intracellular acidification on Kir currents expressed in cultured OLs derived from optic nerves of 12-day-old rats. Unexpectedly, Kir currents in OLs (2-4 DIV) did not change within the pH range of 8.0-5.0, as observed when using standard whole-cell voltage-clamp recording or when preserving cytoplasmic components with the perforated patch-clamp technique. In contrast, low pH inhibited astrocyte Kir currents, which was consistent with the involvement of the Kir4.1 subunit. The H + -insensitivity expressed in OL Kir channels was not intrinsic because Kir cloning showed no difference in the sequence reported for the Kir4.1, Kir2.1, or Kir5.1 subunits. Moreover, when Kir channels were heterologously expressed in Xenopus oocytes they behaved as expected in their general properties and sensitivity to H + . It is therefore concluded that Kir channel H + -sensitivity in OLs is modulated through an extrinsic mechanism, probably by association with a modulatory component or by posttranslational modifications.

Vertebrate rod photoreceptors express both BK and IK calcium-activated potassium channels, but only BK channels are involved in receptor potential regulation.

PubMed

Pelucchi, Bruna; Grimaldi, Annalisa; Moriondo, Andrea

2008-01-01

In salamander rods, Ca(2+)-activated K(+) current (I(KCa)) provides an effective "clamp" of the dark membrane potential to its normal resting level. By a combination of electrophysiological, pharmacological, and immunohistochemical approaches, we show that salamander rods functionally express large-conductance Ca(2+)- and voltage-dependent potassium (BK) channel and intermediate-conductance Ca(2+)-dependent potassium (IK) channel, but not small-conductance Ca(2+)-dependent potassium channel (SK) subtypes. Application of 100 nM iberiotoxin and 100 nM clotrimazole reduced net I(KCa) to 36% and 63%, respectively, whereas the current was unaffected by application of 1 microM apamin. Consistently, anti- SK1, -SK2, and -SK3 antibodies were unable to stain rod photoreceptors, whereas both anti-BK and -SK4/ IK1 antibodies heavily stained the ellipsoid region of the inner segments of the rods. Moreover, by using current-clamp experiments, it was clearly seen that the strong clamping effect of the total I(KCa) was lost when IbTx, but not CLTZ, was applied to the bath. This behavior strongly suggests that of BK and IK channels, only the former are responsible for the clamping effect on the photoreceptor membrane potential.

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.

The Distributions of Voltage-Gated K+ current Subtypes in Different Cell Sizes from Adult Mouse Dorsal Root Ganglia.

PubMed

Sheng, Anqi; Hong, Jiangru; Zhang, Lulu; Zhang, Yan; Zhang, Guangqin

2018-03-29

Voltage-gated K + (K V ) currents play a crucial role in regulating pain by controlling neuronal excitability, and are divided into transient A-type currents (I A ) and delayed rectifier currents (I K ). The dorsal root ganglion (DRG) neurons are heterogeneous and the subtypes of K V currents display different levels in distinct cell sizes. To observe correlations of the subtypes of K V currents with DRG cell sizes, K V currents were recorded by whole-cell patch clamp in freshly isolated mouse DRG neurons. Results showed that I A occupied a high proportion in K V currents in medium- and large-diameter DRG neurons, whereas I K possessed a larger proportion of K V currents in small-diameter DRG neurons. A lower correlation was found between the proportion of I A or I K in K V currents and cell sizes. These data suggest that I A channels are mainly expressed in medium and large cells and I K channels are predominantly expressed in small cells.

Pb2+ Modulates Ca2+ Membrane Permeability In Paramecium

NASA Astrophysics Data System (ADS)

Bernal-Martínez, Juan; Ortega Soto, Arturo

2004-09-01

Intracellular recording experiments in current clamp configuration were done to evaluate whether Pb2+ modulates ionic membrane permeability in the fresh water Paramecium tetraurelia. It was found that Pb2+ triggers in a dose-dependent manner, a burst of spontaneous action potentials followed by a robust and sustained after hyper-polarization. In addition, Pb2+ increased the frequency of firing the spontaneous Ca2+-Action Potential and also, the duration of Ca2+-Action Potential, in a dose and reversibly-dependent manner. These results suggest that Pb2+ increases calcium membrane permeability of Paramecium and probably activates a calcium-dependent-potassium conductance in the ciliate.

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.

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

High quality cord blood banking is feasible with delayed clamping practices. The eight-year experience and current status of the national Swedish Cord Blood Bank.

PubMed

Frändberg, Sofia; Waldner, Berit; Konar, Jan; Rydberg, Lennart; Fasth, Anders; Holgersson, Jan

2016-09-01

The National Swedish Cord Blood Bank (NS-CBB) is altruistic and publicly funded. Herein we describe the status of the bank and the impact of delayed versus early clamping on cell number and volume. Cord Blood Units (CBUs) were collected at two University Hospitals in Sweden. Collected volume and nucleated cell content (TNC) were investigated in 146 consecutive Cord Blood (CB) collections sampled during the first quarter of 2012 and in 162 consecutive CB collections done in the first quarter of 2013, before and after clamping practices were changed from immediate to late (60 s) clamping. NS-CBB now holds close to 5000 units whereof 30 % are from non-Caucasian or mixed origins. Delayed clamping had no major effect on collection efficiency. The volume collected was slightly reduced (mean difference, 8.1 ml; 95 % CI, 1.3-15.0 ml; p = 0.02), while cell recovery was not (p = 0.1). The proportion of CBUs that met initial total TNC banking criteria was 60 % using a TNC threshold of 12.5 × 10(8), and 47 % using a threshold of 15 × 10(8) for the early clamping group and 52 and 37 % in the late clamping group. Following implementation of delayed clamping practices at NS-CBB; close to 40 % of the collections in the late clamping group still met the high TNC banking threshold and were eligible for banking, implicating that that cord blood banking is feasible with delayed clamping practices.

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.

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

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.

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.

Comparison of two Chinese medical herbs, Huangbai and Qianniuzi, on influence of short circuit current across the rat intestinal epithelia.

PubMed

Tsai, Jong-Chang; Tsai, Shuli; Chang, Weng-Cheng

2004-07-01

Huangbai (Phellodendron spec.) and Qianniuzi (Pharbitis spec.) are two traditional Chinese medical herbs used for anti-diarrheal and laxative agents, respectively. Ethanol and water extracts of these two herbs were prepared and effects of the extracts on ion transport of the rat intestinal epithelia were studied. For measuring changes of the short circuit current across the epithelia, the rat intestinal epithelia were mounted in the Ussing chamber and attached with voltage/current clamp. The intestinal epithelia were firstly activated by serosal administration of 5 microM forskolin. As current raised and being stable, extracts of these herbs were added, respectively, and changes in the short circuit current were recorded. Ethanol extract of Huangbai attenuated the current increment; on the contrary, ethanol extract of Qianniuzi augmented the current increment additionally. Water extracts of the two herbs showed minor effects on the current in comparison to ethanol extracts. The results provide evidences to reveal the pharmacological mechanism of the two Chinese medical herbs on the intestinal tissue.

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

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.

[Analysis of brain hemometabolism behavior during carotid endarterectomy with temporary clamping.].

PubMed

Duval Neto, Gastão Fernandes; Niencheski, Augusto H

2004-04-01

Carotid endarterectomy with temporary clamping changes cerebral blood flow and cerebral metabolic oxygen demand ratio with consequent oligemic hypoxia or hemometabolic uncoupling. This study aimed at identifying changes in brain hemometabolism, evaluated through changes in oxyhemoglobin saturation in internal jugular vein bulb (SvjO2) during carotid endarterectomy with clamping, and at correlating these changes with potentially interfering factors, mainly end tidal CO2 pressure (P ET CO2) and cerebral perfusion pressure (CPP). Sixteen patients with unilateral carotid stenotic disease scheduled to carotid endarterectomy with carotid arterial clamping were enrolled in this study. Parameters including internal jugular bulb oxyhemoglobin saturation, stump pressure and end tidal CO2 pressure were measured at the following moments: M1 - pre-clamping; M2 - 3 minutes after clamping; M3 - pre-unclamping; M4 - post-unclamping). The comparison among SvjO2 (%, mean +/- SD) in all studied periods has shown differences between those recorded in moments M1 (52.25 +/- 7.87) and M2 (47.43 +/- 9.19). This initial decrease stabilized during temporary clamping, showing decrease in the comparison between M2 and M3 (46.56 +/- 9.25), without statistical significance (p = ns). At post-unclamping, M4 (47.68 +/- 9.12), SvjO2 was increased as compared to M2 and M3 clamping stages, however it was still lower than that of pre-clamping stage M1.(M4 x M1 - p < 0.04) This SvjO2 decrease was followed by significant cerebral perfusion pressure (stump pressure) decrease. Factors influencing this brain hemometabolic uncoupling trend were correlated to P ET CO2. The comparison between CPP and SvjO2 showed weak correlation devoid of statistical significance. In the conditions of our study, SvjO2 measurement is a fast and effective way of clinically monitoring changes in CBF/CMRO2 ratio. Temporary carotid clamping implies in a trend towards brain hemometabolic uncoupling and, as a consequence, to oligemic ischemia; cerebral perfusion pressure does not assesses brain hemometabolic status (CBF and CMRO2 ratio); hypocapnia, may lead to brain hemometabolic uncoupling; P ET CO2 monitoring is an innocuous and efficient way to indirectly monitor PaCO2 preventing inadvertent hypocapnia and its deleterious effects on CBF/CMRO2 ratio during temporary carotid clamping.

Pharmacological modulation of the voltage-gated neuronal Kv7/KCNQ/M-channel alters the intrinsic excitability and synaptic responses of pyramidal neurons in rat prefrontal cortex slices.

PubMed

Peng, Hui; Bian, Xi-Ling; Ma, Fu-Cui; Wang, Ke-Wei

2017-09-01

The prefrontal cortex (PFC) critical for higher cognition is implicated in neuropsychiatric diseases, such as Alzheimer's disease, depression and schizophrenia. The voltage-activated Kv7/KCNQ/M-channel or M-current modulates the neuronal excitability that defines the fundamental mechanism of brain function. However, whether M-current functions to regulate the excitability of PFC neurons remains elusive. In this study, we recorded the native M-current from PFC layer V pyramidal neurons in rat brain slices and showed that it modulated the intrinsic excitability and synaptic responses of PFC pyramidal neurons. Application of a specific M-channel blocker XE991 (40 μmol/L) or opener retigabine (10 μmol/L) resulted in inhibition or activation of M-current, respectively. In the current-clamp recordings, inhibition of M-current was evidenced by the increased average spike frequency and the reduced first inter-spike interval (ISI), spike onset latency and fast afterhyperpolarization (fAHP), whereas activation of M-current caused opposite responses. Furthermore, inhibition of M-current significantly increased the amplitude of excitatory postsynaptic potentials (EPSPs) and depolarized the resting membrane potential (RMP) without affecting the miniature EPSC (mEPSC) frequency. These data demonstrate that voltage-gated neuronal Kv7/KCNQ/M-current modulates the excitability and synaptic transmission of PFC neurons, suggesting that pharmacological modulation of M-current in the PFC may exert beneficial effects on cognitive deficits implicated in the pathophysiology of neuropsychiatric disorders.

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.

Analysis of the Effects of Calcium or Magnesium on Voltage-Clamp Currents in Perfused Squid Axons Bathed in Solutions of High Potassium

PubMed Central

Rojas, Eduardo; Taylor, Robert E.; Atwater, Illani; Bezanilla, Francisco

1969-01-01

Isolated axons from the squid, Dosidicus gigas, were internally perfused with potassium fluoride solutions. Membrane currents were measured following step changes of membrane potential in a voltage-clamp arrangement with external isosmotic solution changes in the order: potassium-free artificial seawater; potassium chloride; potassium chloride containing 10, 25, 40 or 50, mM calcium or magnesium; and potassium-free artificial seawater. The following results suggest that the currents measured under voltage clamp with potassium outside and inside can be separated into two components and that one of them, the predominant one, is carried through the potassium system. (a) Outward currents in isosmotic potassium were strongly and reversibly reduced by tetraethylammonium chloride. (b) Without calcium or magnesium a progressive increase in the nontime-dependent component of the currents (leakage) occurred. (c) The restoration of calcium or magnesium within 15–30 min decreases this leakage. (d) With 50 mM divalent ions the steady-state current-voltage curve was nonlinear with negative resistance as observed in intact axons in isosmotic potassium. (e) The time-dependent components of the membrane currents were not clearly affected by calcium or magnesium. These results show a strong dependence of the leakage currents on external calcium or magnesium concentration but provide no support for the involvement of calcium or magnesium in the kinetics of the potassium system. PMID:5823216

Analysis of the effects of calcium or magnesium on voltage-clamp currents in perfused squid axons bathed in solutions of high potassium.

PubMed

Rojas, E; Taylor, R E; Atwater, I; Bezanilla, F

1969-10-01

Isolated axons from the squid, Dosidicus gigas, were internally perfused with potassium fluoride solutions. Membrane currents were measured following step changes of membrane potential in a voltage-clamp arrangement with external isosmotic solution changes in the order: potassium-free artificial seawater; potassium chloride; potassium chloride containing 10, 25, 40 or 50, mM calcium or magnesium; and potassium-free artificial seawater. The following results suggest that the currents measured under voltage clamp with potassium outside and inside can be separated into two components and that one of them, the predominant one, is carried through the potassium system. (a) Outward currents in isosmotic potassium were strongly and reversibly reduced by tetraethylammonium chloride. (b) Without calcium or magnesium a progressive increase in the nontime-dependent component of the currents (leakage) occurred. (c) The restoration of calcium or magnesium within 15-30 min decreases this leakage. (d) With 50 mM divalent ions the steady-state current-voltage curve was nonlinear with negative resistance as observed in intact axons in isosmotic potassium. (e) The time-dependent components of the membrane currents were not clearly affected by calcium or magnesium. These results show a strong dependence of the leakage currents on external calcium or magnesium concentration but provide no support for the involvement of calcium or magnesium in the kinetics of the potassium system.

Sodium influxes in internally perfused squid giant axon during voltage clamp.

PubMed

Atwater, I; Bezanilla, F; Rojas, E

1969-05-01

1. An experimental method for measuring ionic influxes during voltage clamp in the giant axon of Dosidicus is described; the technique combines intracellular perfusion with a method for controlling membrane potential.2. Sodium influx determinations were carried out while applying rectangular pulses of membrane depolarization. The ratio ;measured sodium influx/computed ionic flux during the early current' is 0.92 +/- 0.12.3. Plots of measured sodium influx and computed ionic flux during the early current against membrane potential are very similar. There was evidence that the membrane potential at which the sodium influx vanishes is the potential at which the early current reverses.

Hypoxia sensitivity of a voltage-gated potassium current in porcine intrapulmonary vein smooth muscle cells.

PubMed

Dospinescu, Ciprian; Widmer, Hélène; Rowe, Iain; Wainwright, Cherry; Cruickshank, Stuart F

2012-09-01

Hypoxia contracts the pulmonary vein, but the underlying cellular effectors remain unclear. Utilizing contractile studies and whole cell patch-clamp electrophysiology, we report for the first time a hypoxia-sensitive K(+) current in porcine pulmonary vein smooth muscle cells (PVSMC). Hypoxia induced a transient contractile response that was 56 ± 7% of the control response (80 mM KCl). This contraction required extracellular Ca(2+) and was sensitive to Ca(2+) channel blockade. Blockade of K(+) channels by tetraethylammonium chloride (TEA) or 4-aminopyridine (4-AP) reversibly inhibited the hypoxia-mediated contraction. Single-isolated PVSMC (typically 159.1 ± 2.3 μm long) had mean resting membrane potentials (RMP) of -36 ± 4 mV with a mean membrane capacitance of 108 ± 3.5 pF. Whole cell patch-clamp recordings identified a rapidly activating, partially inactivating K(+) current (I(KH)) that was hypoxia, TEA, and 4-AP sensitive. I(KH) was insensitive to Penitrem A or glyburide in PVSMC and had a time to peak of 14.4 ± 3.3 ms and recovered in 67 ms following inactivation at +80 mV. Peak window current was -32 mV, suggesting that I(KH) may contribute to PVSMC RMP. The molecular identity of the potassium channel is not clear. However, RT-PCR, using porcine pulmonary artery and vein samples, identified Kv(1.5), Kv(2.1), and BK, with all three being more abundant in the PV. Both artery and vein expressed STREX, a highly conserved and hypoxia-sensitive BK channel variant. Taken together, our data support the hypothesis that hypoxic inhibition of I(KH) would contribute to hypoxic-induced contraction in PVSMC.

Voltage-clamp analysis of membrane currents and excitation-contraction coupling in a crustacean muscle.

PubMed

Weiss, T; Erxleben, C; Rathmayer, W

2001-01-01

A single fibre preparation from the extensor muscle of a marine isopod crustacean is described which allows the analysis of membrane currents and simultaneously recorded contractions under two-electrode voltage-clamp conditions. We show that there are three main depolarisation-gated currents, two are outward and carried by K+, the third is an inward Ca2+ current, I(Ca). Normally, the K+ currents which can be isolated by using K+ channel blockers, mask I(Ca). I(Ca) activates at potentials more positive than -40 mV, is maximal around 0 mV, and shows strong inactivation at higher depolarisation. Inactivation depends on current rather than voltage. Ba2+, Sr2+ and Mg2+ can substitute for Ca2+. Ba2+ currents are about 80% larger than Ca2+ currents and inactivate little. The properties of I(Ca) characterise it as a high threshold L-type current. The outward current consists primarily of a fast, transient A current, I(K(A)) and a maintained, delayed rectifier current, I(K(V)). In some fibres, a small Ca2+-dependent K+ current is also present. I(K(A)) activates fast at depolarisation above -45 mV, shows pronounced inactivation and is almost completely inactivated at holding potentials more positive than -40 mV. I(K(A)) is half-maximally blocked by 70 microM 4-aminopyridine (4-AP), and 70 mM tetraethylammonium (TEA). I(K(V)) activates more slowly, at about -30 mV, and shows no inactivation. It is half-maximally blocked by 2 mM TEA but rather insensitive to 4-AP. Physiologically, the two K+ currents prevent all-or-nothing action potentials and determine the graded amplitude of active electrical responses and associated contractions. Tension development depends on and is correlated with depolarisation-induced Ca2+ influx mediated by I(Ca). The voltage dependence of peak tension corresponds directly to the voltage dependence of the integrated I(Ca). The threshold potential for contraction is at about -38 mV. Peak tension increases with increasing voltage steps, reaches maximum at around 0 mV, and declines with further depolarisation.

The computational structural mechanics testbed architecture. Volume 4: The global-database manager GAL-DBM

NASA Technical Reports Server (NTRS)

Wright, Mary A.; Regelbrugge, Marc E.; Felippa, Carlos A.

1989-01-01

This is the fourth of a set of five volumes which describe the software architecture for the Computational Structural Mechanics Testbed. Derived from NICE, an integrated software system developed at Lockheed Palo Alto Research Laboratory, the architecture is composed of the command language CLAMP, the command language interpreter CLIP, and the data manager GAL. Volumes 1, 2, and 3 (NASA CR's 178384, 178385, and 178386, respectively) describe CLAMP and CLIP and the CLIP-processor interface. Volumes 4 and 5 (NASA CR's 178387 and 178388, respectively) describe GAL and its low-level I/O. CLAMP, an acronym for Command Language for Applied Mechanics Processors, is designed to control the flow of execution of processors written for NICE. Volume 4 describes the nominal-record data management component of the NICE software. It is intended for all users.

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.

Tribologic analyses of a self-mated aluminium contact used for overhead transmission lines

NASA Astrophysics Data System (ADS)

Steier, V. Franco

2017-05-01

The lifetime of aluminium components is often limited to their poor wear resistance. One example for such aluminium applications are overhead transmission lines. The sore points of these lines are the segments where the aluminium conductors are fixed to the line supports. The fixation is commonly realized via aluminium suspension clamps. Here, a superposition of different loads like traction and bending stresses, clamping forces and different types of wear occurs. To investigate the wear behaviour in these peculiar points, tribologic model tests were carried out. Within the tests, overhead conductor wires and aluminium plates, extracted from suspension clamps were reciprocally slid against aluminium plates (cylinder-on-plate test). The COF and a wear related parameter were recorded constantly. Subsequently, the loaded surfaces were analysed using confocal laser and electron scanning microscopy as well as energy dispersive X-ray spectroscopy. The investigation detected the formation of an oxidized tribologic layer between both components. The tribolayer, which mayor part adhered on the suspension clamps, was mostly formed from material removed from the conductor wires.

Implementation of a fast 16-Bit dynamic clamp using LabVIEW-RT.

PubMed

Kullmann, Paul H M; Wheeler, Diek W; Beacom, Joshua; Horn, John P

2004-01-01

The dynamic-clamp method provides a powerful electrophysiological tool for creating virtual ionic conductances in living cells and studying their influence on membrane potential. Here we describe G-clamp, a new way to implement a dynamic clamp using the real-time version of the Lab-VIEW programming environment together with a Windows host, an embedded microprocessor that runs a real-time operating system and a multifunction data-acquisition board. The software includes descriptions of a fast voltage-dependent sodium conductance, delayed rectifier, M-type and A-type potassium conductances, and a leak conductance. The system can also read synaptic conductance waveforms from preassembled data files. These virtual conductances can be reliably implemented at speeds < or =43 kHz while simultaneously saving two channels of data with 16-bit precision. G-clamp also includes utilities for measuring current-voltage relations, synaptic strength, and synaptic gain. Taking an approach built on a commercially available software/hardware platform has resulted in a system that is easy to assemble and upgrade. In addition, the graphical programming structure of LabVIEW should make it relatively easy for others to adapt G-clamp for new experimental applications.

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

Self-organised criticality and 1/f noise in single-channel current of voltage-dependent anion channel

NASA Astrophysics Data System (ADS)

Banerjee, J.; Verma, M. K.; Manna, S.; Ghosh, S.

2006-02-01

Noise profile of Voltage Dependent Anion Channel (VDAC) is investigated in open channel state. Single-channel currents through VDAC from mitochondria of rat brain reconstituted into a planar lipid bilayer are recorded under different voltage clamped conditions across the membrane. Power spectrum analysis of current indicates power law noise of 1/f nature. Moreover, this 1/f nature of the open channel noise is seen throughout the range of applied membrane potential from -30 to +30 mV. It is being proposed that 1/f noise in open ion channel arises out of obstruction in the passage of ions across the membrane. The process is recognised as a phenomenon of self-organized criticality (SOC) like sandpile avalanche and other physical systems. Based on SOC it has been theoretically established that the system of ion channel follows power law noise as observed in our experiments. We also show that the first-time return probability of current fluctuations obeys a power law distribution.

Role of the pH in state-dependent blockade of hERG currents

NASA Astrophysics Data System (ADS)

Wang, Yibo; Guo, Jiqing; Perissinotti, Laura L.; Lees-Miller, James; Teng, Guoqi; Durdagi, Serdar; Duff, Henry J.; Noskov, Sergei Yu.

2016-10-01

Mutations that reduce inactivation of the voltage-gated Kv11.1 potassium channel (hERG) reduce binding for a number of blockers. State specific block of the inactivated state of hERG block may increase risks of drug-induced Torsade de pointes. In this study, molecular simulations of dofetilide binding to the previously developed and experimentally validated models of the hERG channel in open and open-inactivated states were combined with voltage-clamp experiments to unravel the mechanism(s) of state-dependent blockade. The computations of the free energy profiles associated with the drug block to its binding pocket in the intra-cavitary site display startling differences in the open and open-inactivated states of the channel. It was also found that drug ionization may play a crucial role in preferential targeting to the open-inactivated state of the pore domain. pH-dependent hERG blockade by dofetilie was studied with patch-clamp recordings. The results show that low pH increases the extent and speed of drug-induced block. Both experimental and computational findings indicate that binding to the open-inactivated state is of key importance to our understanding of the dofetilide’s mode of action.

Aortic Baroreceptors Display Higher Mechanosensitivity than Carotid Baroreceptors.

PubMed

Lau, Eva On-Chai; Lo, Chun-Yin; Yao, Yifei; Mak, Arthur Fuk-Tat; Jiang, Liwen; Huang, Yu; Yao, Xiaoqiang

2016-01-01

Arterial baroreceptors are mechanical sensors that detect blood pressure changes. It has long been suggested that the two arterial baroreceptors, aortic and carotid baroreceptors, have different pressure sensitivities. However, there is no consensus as to which of the arterial baroreceptors are more sensitive to changes in blood pressure. In the present study, we employed independent methods to compare the pressure sensitivity of the two arterial baroreceptors. Firstly, pressure-activated action potential firing was measured by whole-cell current clamp with a high-speed pressure clamp system in primary cultured baroreceptor neurons. The results show that aortic depressor neurons possessed a higher percentage of mechano-sensitive neurons. Furthermore, aortic baroreceptor neurons show a lower pressure threshold than that of carotid baroreceptor neurons. Secondly, uniaxial stretching of baroreceptor neurons, that mimics the forces exerted on blood vessels, elicited a larger increase in intracellular Ca(2+) rise in aortic baroreceptor neurons than in carotid baroreceptor neurons. Thirdly, the pressure-induced action potential firing in the aortic depressor nerve recorded in vivo was also higher. The present study therefore provides for a basic physiological understanding on the pressure sensitivity of the two baroreceptor neurons and suggests that aortic baroreceptors have a higher pressure sensitivity than carotid baroreceptors.

A calibration-free electrode compensation method

PubMed Central

Rossant, Cyrille; Fontaine, Bertrand; Magnusson, Anna K.

2012-01-01

In a single-electrode current-clamp recording, the measured potential includes both the response of the membrane and that of the measuring electrode. The electrode response is traditionally removed using bridge balance, where the response of an ideal resistor representing the electrode is subtracted from the measurement. Because the electrode is not an ideal resistor, this procedure produces capacitive transients in response to fast or discontinuous currents. More sophisticated methods exist, but they all require a preliminary calibration phase, to estimate the properties of the electrode. If these properties change after calibration, the measurements are corrupted. We propose a compensation method that does not require preliminary calibration. Measurements are compensated offline by fitting a model of the neuron and electrode to the trace and subtracting the predicted electrode response. The error criterion is designed to avoid the distortion of compensated traces by spikes. The technique allows electrode properties to be tracked over time and can be extended to arbitrary models of electrode and neuron. We demonstrate the method using biophysical models and whole cell recordings in cortical and brain-stem neurons. PMID:22896724

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.

Altered Ca2+ signaling in skeletal muscle fibers of the R6/2 mouse, a model of Huntington’s disease

PubMed Central

Braubach, Peter; Orynbayev, Murat; Andronache, Zoita; Hering, Tanja; Landwehrmeyer, Georg Bernhard; Lindenberg, Katrin S.

2014-01-01

Huntington’s disease (HD) is caused by an expanded CAG trinucleotide repeat within the gene encoding the protein huntingtin. The resulting elongated glutamine (poly-Q) sequence of mutant huntingtin (mhtt) affects both central neurons and skeletal muscle. Recent reports suggest that ryanodine receptor–based Ca2+ signaling, which is crucial for skeletal muscle excitation–contraction coupling (ECC), is changed by mhtt in HD neurons. Consequently, we searched for alterations of ECC in muscle fibers of the R6/2 mouse, a mouse model of HD. We performed fluorometric recordings of action potentials (APs) and cellular Ca2+ transients on intact isolated toe muscle fibers (musculi interossei), and measured L-type Ca2+ inward currents on internally dialyzed fibers under voltage-clamp conditions. Both APs and AP-triggered Ca2+ transients showed slower kinetics in R6/2 fibers than in fibers from wild-type mice. Ca2+ removal from the myoplasm and Ca2+ release flux from the sarcoplasmic reticulum were characterized using a Ca2+ binding and transport model, which indicated a significant reduction in slow Ca2+ removal activity and Ca2+ release flux both after APs and under voltage-clamp conditions. In addition, the voltage-clamp experiments showed a highly significant decrease in L-type Ca2+ channel conductance. These results indicate profound changes of Ca2+ turnover in skeletal muscle of R6/2 mice and suggest that these changes may be associated with muscle pathology in HD. PMID:25348412

Timing of umbilical cord-clamping and infant anaemia: the role of maternal anaemia.

PubMed

Blouin, Brittany; Penny, Mary E; Maheu-Giroux, Mathieu; Casapía, Martín; Aguilar, Eder; Silva, Hermánn; Creed-Kanashiro, Hilary M; Joseph, Serene A; Gagnon, Anita; Rahme, Elham; Gyorkos, Theresa W

2013-05-01

Evidence from randomized controlled trials has shown that delayed cord-clamping is beneficial to infant iron status. The role of maternal anaemia in this relationship, however, has not been established. To determine the effect of maternal anaemia at delivery on the association between timing of umbilical cord-clamping and infant anaemia at 4 and 8 months of age. A cohort of pregnant women admitted to the labour room of Hospital Iquitos (Iquitos, Peru) and their newborns were recruited into the study during two time periods (18 May to 3 June and 6-20 July 2009). Between the two recruitment periods, the hospital's policy changed from early to delayed umbilical cord-clamping. Maternal haemoglobin levels were measured before delivery, and the time between delivery and cord-clamping was recorded at delivery for the entire cohort. Mother-infant pairs were followed-up at 4 (n = 207) and 8 months (n = 184) post partum. Infant haemoglobin levels were measured at follow-up visits. Data were analysed using logistic regression models. The prevalence of maternal anaemia (Hb <11.0 g/dl) at delivery was 22%. Infant haemoglobin levels at 4 and 8 months of age were 10.4 g/dl and 10.3 g/dl, respectively. Infant haemoglobin levels did not differ significantly between infants born to anaemic mothers and those born to non-anaemic mothers at either 4 or 8 months of age. However, the association between the timing of cord-clamping and infant anaemia was modified by the mother's anaemia status. Significant benefits of delayed cord-clamping in preventing anaemia were found in infants born to anaemic mothers at both 4 months (aOR = 0.59, 95% CI 0.36-0.99) and 8 months (aOR = 0.38, 95% CI 0.19-0.76) of age. The study contributes additional evidence in support of delayed cord-clamping. This intervention is likely to have most public health impact in areas with a high prevalence of anaemia during pregnancy.

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

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

The Semicircular Canal Microphonic

NASA Technical Reports Server (NTRS)

Rabbitt, R. D.; Boyle, R.; Highstein, S. M.; Dalton, Bonnie P. (Technical Monitor)

2002-01-01

Present experiments were designed to quantify the alternating current (AC) component of the semicircular canal microphonic for angular motion stimulation as a function of stimulus frequency and amplitude. The oyster toadfish, Opsanus tau, was used as the experimental model. Calibrated mechanical indentation of the horizontal canal duct was used as a stimulus to generate hair-cell and afferent responses reproducing those present during head rotation. Sensitivity to polarization of the endolymph DC voltage re: perilymph was also investigated. Modulation of endolymph voltage was recorded using conventional glass electrodes and lock-in amplification over the frequency range 0.2-80 Hz. Access to the endolymph for inserting voltage recording and current passing electrodes was obtained by sectioning the anterior canal at its apex and isolating the cut ends in air. For sinusoidal stimulation below approx.10 Hz, the horizontal semicircular canal AC microphonic was nearly independent of stimulus frequency and equal to approximately 4 microV per micron indent (equivalent to approx. 1 microV per deg/s). A saturating nonlinearity decreasing the microphonic gain was present for stimuli exceeding approx.3 micron indent (approx. 12 deg/s angular velocity). The phase was not sensitive to the saturating nonlinearity. The microphonic exhibited a resonance near 30Hz consistent with basolateral current hair cell resonance observed previously in voltage-clamp records from semicircular canal hair cells. The magnitude and phase of the microphonic exhibited sensitivity to endolymphatic polarization consistent with electro-chemical reversal of hair cell transduction currents.

Bipolar square-wave current source for transient electromagnetic systems based on constant shutdown time

NASA Astrophysics Data System (ADS)

Wang, Shilong; Yin, Changchun; Lin, Jun; Yang, Yu; Hu, Xueyan

2016-03-01

Cooperative work of multiple magnetic transmitting sources is a new trend in the development of transient electromagnetic system. The key is the bipolar current waves shutdown, concurrently in the inductive load. In the past, it was difficult to use the constant clamping voltage technique to realize the synchronized shutdown of currents with different peak values. Based on clamping voltage technique, we introduce a new controlling method with constant shutdown time. We use the rising time to control shutdown time and use low voltage power source to control peak current. From the viewpoint of the circuit energy loss, by taking the high-voltage capacitor bypass resistance and the capacitor of the passive snubber circuit into account, we establish the relationship between the rising time and the shutdown time. Since the switch is not ideal, we propose a new method to test the shutdown time by the low voltage, the high voltage and the peak current. Experimental results show that adjustment of the current rising time can precisely control the value of the clamp voltage. When the rising time is fixed, the shutdown time is unchanged. The error for shutdown time deduced from the energy consumption is less than 6%. The new controlling method on current shutdown proposed in this paper can be used in the cooperative work of borehole and ground transmitting system.

Bipolar square-wave current source for transient electromagnetic systems based on constant shutdown time.

PubMed

Wang, Shilong; Yin, Changchun; Lin, Jun; Yang, Yu; Hu, Xueyan

2016-03-01

Cooperative work of multiple magnetic transmitting sources is a new trend in the development of transient electromagnetic system. The key is the bipolar current waves shutdown, concurrently in the inductive load. In the past, it was difficult to use the constant clamping voltage technique to realize the synchronized shutdown of currents with different peak values. Based on clamping voltage technique, we introduce a new controlling method with constant shutdown time. We use the rising time to control shutdown time and use low voltage power source to control peak current. From the viewpoint of the circuit energy loss, by taking the high-voltage capacitor bypass resistance and the capacitor of the passive snubber circuit into account, we establish the relationship between the rising time and the shutdown time. Since the switch is not ideal, we propose a new method to test the shutdown time by the low voltage, the high voltage and the peak current. Experimental results show that adjustment of the current rising time can precisely control the value of the clamp voltage. When the rising time is fixed, the shutdown time is unchanged. The error for shutdown time deduced from the energy consumption is less than 6%. The new controlling method on current shutdown proposed in this paper can be used in the cooperative work of borehole and ground transmitting system.

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.

MACHINING ELIMINATION THROUGH APPLICATION OF THREAD FORMING FASTENERS IN NET SHAPED CAST HOLES

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

Cleaver, Ryan J; Cleaver, Todd H; Talbott, Richard

The ultimate objective of this work was to eliminate approximately 30% of the machining performed in typical automotive engine and transmission plants by using thread forming fasteners in as-cast holes of aluminum and magnesium cast components. The primary issues at the source of engineers reluctance to implementing thread forming fasteners in lightweight castings are: * Little proof of consistency of clamp load vs. input torque in either aluminum or magnesium castings. * No known data to understand the effect on consistency of clamp load as casting dies wear. The clamp load consistency concern is founded in the fact that amore » portion of the input torque used to create clamp load is also used to create threads. The torque used for thread forming may not be consistent due to variations in casting material, hole size and shape due to tooling wear and process variation (thermal and mechanical). There is little data available to understand the magnitude of this concern or to form the basis of potential solutions if the range of clamp load variation is very high (> +/- 30%). The range of variation that can be expected in as-cast hole size and shape over the full life cycle of a high pressure die casting die was established in previous work completed by Pacific Northwest National Laboratory, (PNNL). This established range of variation was captured in a set of 12 cast bosses by designing core pins at the size and draft angles identified in the sited previous work. The cast bosses were cut into nuts that could be used in the Ford Fastener Laboratory test-cell to measure clamp load when a thread forming fastener was driven into a cast nut. There were two sets of experiments run. First, a series of cast aluminum nuts were made reflecting the range of shape and size variations to be expected over the life cycle of a die casting die. Taptite thread forming fasteners, (a widely used thread forming fastener suitable for aluminum applications), were driven into the various cored, as-cast nuts at a constant input torque and resulting clamp loads were recorded continuously. The clamp load data was used to determine the range of clamp loads to be expected. The bolts were driven to failure. The clamp load corresponding to the target input of 18.5 Nm was recorded for each fastener. In a like fashion, a second set of experiments were run with cast magnesium nuts and ALtracs thread forming fasteners, (a widely used thread forming fastener suitable for magnesium applications). Again all clamp loads were recorded and analyzed similarly to the Taptites in aluminum cast nuts. Results from previous work performed on the same test cell for a Battelle project using standard M8 bolts into standard M8 nuts were included as a comparator for a standard bolt and nut application. The results for the thread forming fasteners in aluminum cast holes were well within industry expectations of +/- 30% for out of the box and robustness range testing. The results for the dry and lubed extreme conditions were only slightly higher than industry expectations at +/- 35.6%. However, when compared to the actual Battelle results (+/- 40%) for a standard bolt and nut the tread forming fasteners performed slightly better. The results for the thread forming fasteners in magnesium cast holes were all well within industry expectations of +/- 30% for all three conditions. The robustness range (.05mm larger and smaller holes than the expected wear pattern of a die casting die at full life cycle) results also fell within the industry expectations for standard threaded fasteners. These results were very encouraging. It was concluded that this work showed that clamp load variation with thread forming fasteners is consistent with industry expectations for standard steel bolts and nuts at +/- 30%. There does not appear to be any significant increase in clamp load variation due to the application of thread forming fasteners in as-cast holes of aluminum or magnesium over the effective life of a die casting mold. The fully implemented potential benefit of thread forming fasteners in as-cast holes of aluminum and magnesium is estimated to be 6 trillion Btu per year for North America. Economic benefit is estimated to be nearly $800 million per year. Environmental benefits and quality improvements will also result from full implementation of this technology.« less

Two forms of electrical resonance at theta frequencies, generated by M-current, h-current and persistent Na+ current in rat hippocampal pyramidal cells

PubMed Central

Hu, Hua; Vervaeke, Koen; Storm, Johan F

2002-01-01

Coherent network oscillations in the brain are correlated with different behavioural states. Intrinsic resonance properties of neurons provide a basis for such oscillations. In the hippocampus, CA1 pyramidal neurons show resonance at theta (θ) frequencies (2-7 Hz). To study the mechanisms underlying θ-resonance, we performed whole-cell recordings from CA1 pyramidal cells (n = 73) in rat hippocampal slices. Oscillating current injections at different frequencies (ZAP protocol), revealed clear resonance with peak impedance at 2-5 Hz at ≈33 °C (increasing to ≈7 Hz at ≈38 °C). The θ-resonance showed a U-shaped voltage dependence, being strong at subthreshold, depolarized (≈-60 mV) and hyperpolarized (≈-80 mV) potentials, but weaker near the resting potential (-72 mV). Voltage clamp experiments revealed three non-inactivating currents operating in the subthresold voltage range: (1) M-current (IM), which activated positive to -65 mV and was blocked by the M/KCNQ channel blocker XE991 (10 μm); (2) h-current (Ih), which activated negative to -65 mV and was blocked by the h/HCN channel blocker ZD7288 (10 μm); and (3) a persistent Na+ current (INaP), which activated positive to -65 mV and was blocked by tetrodotoxin (TTX, 1 μm). In current clamp, XE991 or TTX suppressed the resonance at depolarized, but not hyperpolarized membrane potentials, whereas ZD7288 abolished the resonance only at hyperpolarized potentials. We conclude that these cells show two forms of θ-resonance: ‘M-resonance’ generated by the M-current and persistent Na+ current in depolarized cells, and ‘H-resonance’ generated by the h-current in hyperpolarized cells. Computer simulations supported this interpretation. These results suggest a novel function for M/KCNQ channels in the brain: to facilitate neuronal resonance and network oscillations in cortical neurons, thus providing a basis for an oscillation-based neural code. PMID:12482886

Voltage and Current Clamp Transients with Membrane Dielectric Loss

PubMed Central

Fitzhugh, R.; Cole, K. S.

1973-01-01

Transient responses of a space-clamped squid axon membrane to step changes of voltage or current are often approximated by exponential functions of time, corresponding to a series resistance and a membrane capacity of 1.0 μF/cm2. Curtis and Cole (1938, J. Gen. Physiol. 21:757) found, however, that the membrane had a constant phase angle impedance z = z1(jωτ)-α, with a mean α = 0.85. (α = 1.0 for an ideal capacitor; α < 1.0 may represent dielectric loss.) This result is supported by more recently published experimental data. For comparison with experiments, we have computed functions expressing voltage and current transients with constant phase angle capacitance, a parallel leakage conductance, and a series resistance, at nine values of α from 0.5 to 1.0. A series in powers of tα provided a good approximation for short times; one in powers of t-α, for long times; for intermediate times, a rational approximation matching both series for a finite number of terms was used. These computations may help in determining experimental series resistances and parallel leakage conductances from membrane voltage or current clamp data. PMID:4754194

Electrophysiological Recordings from Lobula Plate Tangential Cells in Drosophila.

PubMed

Mauss, Alex S; Borst, Alexander

2016-01-01

Drosophila has emerged as an important model organism for the study of the neural basis of behavior. Its main asset is the experimental accessibility of identified neurons by genetic manipulation and physiological recordings. Drosophila therefore offers the opportunity to reach an integrative understanding of the development and neural underpinnings of behavior at all processing stages, from sensing to motor control, in a single species. Here, we will provide an account of the procedures involved in recording the electrical potential of individual neurons in the visual system of adult Drosophila using the whole-cell patch-clamp method. To this end, animals are fixed to a holder and mounted below a recording chamber. The head capsule is cut open and the glial sheath covering the brain is ruptured by a combination of shearing and enzymatic digest. Neuronal somata are thus exposed and targeted by low-resistance patch electrodes. After formation of a high resistance seal, electrical access to the cell is gained by small current pulses and suction. Stable recordings of large neurons are feasible for >1 h and can be combined with controlled visual stimulation as well as genetic and pharmacological manipulation of upstream circuit elements to infer circuit function in great detail.

Developing an evidence-based nursing protocol on wound drain management for total joint arthroplasty.

PubMed

Tsang, Lap Fung

2015-05-01

Although various drains have long been used for many years in total joint replacement, there is a paucity of evidence for the benefit of drain applications. Evidence suggests inconsistent practice in the use of drainage systems, whether intermittently applying suction or free of suction in the application of drainage systems, as well as the optimal timing for wound drain removal. It aimed to perform a systematic review to develop an evidence-based nursing protocol to manage wound drainage following total joint arthroplasty. A comprehensive systematic review of available evidence up to 2013. Searches of the EMBASE, Cochrane library, CINAHL, Medline electronic databases and an internet search by Yahoo and Google engine returned 2840 records, of which 11 met the inclusion criteria for this review. A further two papers were obtained through scanning the reference lists of those articles included from the initial literature search. Different clamping times were retrieved from the literature. A protocol was adapted for clinical application according to the summary of the retrieved information. It is suggested that clamping is performed 1 h after the insertion of suction drains post-operatively in the operating theatre. Wound drains should be clamped for 1 h if blood loss is more than 600 ml in 6 h in first 24 h. Wound drains should be clamped for 1 h if blood loss is more than 800 ml in 8 h thereafter. It is suggested that the drainage reservoir bottle should be mark and findings recorded in line with the principle of drain clamping. This means that the amount of drainage is measured and recorded every 6 h in first 24 h and every 8 h thereafter. It is suggested that wound drains should be remove before 48 h after TJR. If blood loss is less than 50 ml in past 6 h or less than 70 ml in past 8 h, the drain should be remove and the wound site should be monitored closely. This paper has guided nurses to develop an evidence-based protocol to improve patient care on wound drain management. Further study is necessary to evaluate the effectiveness of the protocol. Copyright © 2014 Elsevier Ltd. All rights reserved.

Nanopore Logic Operation with DNA to RNA Transcription in a Droplet System.

PubMed

Ohara, Masayuki; Takinoue, Masahiro; Kawano, Ryuji

2017-07-21

This paper describes an AND logic operation with amplification and transcription from DNA to RNA, using T7 RNA polymerase. All four operations, (0 0) to (1 1), with an enzyme reaction can be performed simultaneously, using four-droplet devices that are directly connected to a patch-clamp amplifier. The output RNA molecule is detected using a biological nanopore with single-molecule translocation. Channel current recordings can be obtained using the enzyme solution. The integration of DNA logic gates into electrochemical devices is necessary to obtain output information in a human-recognizable form. Our method will be useful for rapid and confined DNA computing applications, including the development of programmable diagnostic devices.

TRPM4 non-selective cation channels influence action potentials in rabbit Purkinje fibres.

PubMed

Hof, Thomas; Sallé, Laurent; Coulbault, Laurent; Richer, Romain; Alexandre, Joachim; Rouet, René; Manrique, Alain; Guinamard, Romain

2016-01-15

The transient receptor potential melastatin 4 (TRPM4) inhibitor 9-phenanthrol reduces action potential duration in rabbit Purkinje fibres but not in ventricle. TRPM4-like single channel activity is observed in isolated rabbit Purkinje cells but not in ventricular cells. The TRPM4-like current develops during the notch and early repolarization phases of the action potential in Purkinje cells. Transient receptor potential melastatin 4 (TRPM4) Ca(2+)-activated non-selective cation channel activity has been recorded in cardiomyocytes and sinus node cells from mammals. In addition, TRPM4 gene mutations are associated with human diseases of cardiac conduction, suggesting that TRPM4 plays a role in this aspect of cardiac function. Here we evaluate the TRPM4 contribution to cardiac electrophysiology of Purkinje fibres. Ventricular strips with Purkinje fibres were isolated from rabbit hearts. Intracellular microelectrodes recorded Purkinje fibre activity and the TRPM4 inhibitor 9-phenanthrol was applied to unmask potential TRPM4 contributions to the action potential. 9-Phenanthrol reduced action potential duration measured at the point of 50 and 90% repolarization with an EC50 of 32.8 and 36.1×10(-6) mol l(-1), respectively, but did not modulate ventricular action potentials. Inside-out patch-clamp recordings were used to monitor TRPM4 activity in isolated Purkinje cells. TRPM4-like single channel activity (conductance = 23.8 pS; equal permeability for Na(+) and K(+); sensitivity to voltage, Ca(2+) and 9-phenanthrol) was observed in 43% of patches from Purkinje cells but not from ventricular cells (0/16). Action potential clamp experiments performed in the whole-cell configuration revealed a transient inward 9-phenanthrol-sensitive current (peak density = -0.65 ± 0.15 pA pF(-1); n = 5) during the plateau phases of the Purkinje fibre action potential. These results show that TRPM4 influences action potential characteristics in rabbit Purkinje fibres and thus could modulate cardiac conduction and be involved in triggering arrhythmias. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Sodium influxes in internally perfused squid giant axon during voltage clamp

PubMed Central

Atwater, I.; Bezanilla, F.; Rojas, E.

1969-01-01

1. An experimental method for measuring ionic influxes during voltage clamp in the giant axon of Dosidicus is described; the technique combines intracellular perfusion with a method for controlling membrane potential. 2. Sodium influx determinations were carried out while applying rectangular pulses of membrane depolarization. The ratio `measured sodium influx/computed ionic flux during the early current' is 0·92 ± 0·12. 3. Plots of measured sodium influx and computed ionic flux during the early current against membrane potential are very similar. There was evidence that the membrane potential at which the sodium influx vanishes is the potential at which the early current reverses. PMID:5767887

Rapid communication between neurons and astrocytes in primary cortical cultures.

PubMed

Murphy, T H; Blatter, L A; Wier, W G; Baraban, J M

1993-06-01

The identification of neurotransmitter receptors and voltage-sensitive ion channels on astrocytes (reviewed by Barres, 1991) has renewed interest in how these cells respond to neuronal activity. To investigate the physiology of neuron astrocyte signaling, we have employed primary cortical cultures that contain both neuronal and glial cells. As the neurons in these cultures exhibit synchronous spontaneous synaptic activity, we have used both calcium imaging and whole-cell recording techniques to identify physiological activity in astrocytes related to neuronal activity. Whole-cell voltage-clamp records from astrocytes revealed rapid inward currents that coincide with bursts of electrical activity in neighboring neurons. Calcium imaging studies demonstrate that these currents in astrocytes are not always associated with slowly propagating calcium waves. Inclusion of the dye Lucifer yellow within patch pipettes confirmed that astrocytes are extensively coupled to each other but not to adjacent neurons, indicating that the currents observed are not due to gap junction connections between these cell types. These currents do not reflect widespread diffusion of glutamate or potassium released during neuronal activity since a population of small, round, multipolar presumed glial cells that are not dye coupled to adjacent cells did not display electrical currents coincident with neuronal firing, even though they respond to locally applied glutamate and potassium. These findings indicate that, in addition to the relatively slow signaling conveyed by calcium waves, astrocytes also display rapid electrical responses to neuronal activity.

[The effect of niflumic acid on gamma aminobutyric acid activated current in DRG neurons].

PubMed

Li, Li; Li, Jing; Ma, Ke-Tao; Cheng, Hong-Ju; Zhao, Lei; Wang, Yang; Si, Jun-Qiang

2013-01-01

To explore the modulatory effect of niflumic acid (NFA) on gamma aminobutyric acid (GABA)-activated currents of dorsal root ganglion (DRG) neurons in rat. The whole-cell patch-clamp technique was used to record the NFA- and GABA-activated currents in neurons freshly dissociated from rat DRG neurons. Application of NFA(0.1 - 100 micromol/L) could induce concentration-dependent outward currents in some cells (21/48,43.75%), and GABA (0.1 - 100 micromol/L) could induce concentration-dependent inward currents in some cells(150/159,94.32%). NFA-(100 micromol/L) and GABA-(100 micromol/L) activated currents were (0.27 +/- 0.06) nA (n = 12) and (1.29 +/- 0.72) nA (n = 53) respectively. However, pre-application of NFA (0.1 - 100 micromol/L) could inhibit the GABA-activated inward current which was identified to be GABAA receptor-mediated current. The inhibitory effects of NFA were concentration-dependent. NFA could not alter the EC50 (about 30 micromol/L) and inverse potential (about -10 mV) of GABA-activated current (P > 0.05). Pre-application of NFA exerts a more strong inhibitory effect on the peak value of GABA-activated current.

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

Verification of the windings axial clamping forces for high voltage power transformers by using passively mode-locked fiber lasers

NASA Astrophysics Data System (ADS)

Şchiopu, IonuÅ£ Romeo; ǎgulinescu, Andrei, Dr; Iordǎnescu, Raluca; Marinescu, Andrei

2015-02-01

The current paper describes an optoelectronic method for direct monitoring of the axial clamping forces both in static and in dynamic duty. As advantages of this method we can state that it can be applied both to new and refurbished transformers without performing constructive changes or affecting in any way the transformer safety in operation. For monitoring the axial clamping forces for high-voltage (HV) power transformers, we use an optical fiber that we integrate into the laser cavity of a passively mode-locked fiber laser (PMFL). To each axial clamp corresponds a solitonic optical spectrum that is changed at the periodical passing of the fundamental soliton pulse through the sensitive fiber inside the transformer. Moreover, as a specific characteristic, the laser stability is unique for each set of axial clamping forces. Other important advantages of using an optical fiber as compared to the classical approach in which electronic sensors are used consist in the good reliability and insulator properties of the optical fiber, avoiding any risk of fire or damage of the transformer.

Membrane Properties and the Balance between Excitation and Inhibition Control Gamma-Frequency Oscillations Arising from Feedback Inhibition

PubMed Central

Economo, Michael N.; White, John A.

2012-01-01

Computational studies as well as in vivo and in vitro results have shown that many cortical neurons fire in a highly irregular manner and at low average firing rates. These patterns seem to persist even when highly rhythmic signals are recorded by local field potential electrodes or other methods that quantify the summed behavior of a local population. Models of the 30–80 Hz gamma rhythm in which network oscillations arise through ‘stochastic synchrony’ capture the variability observed in the spike output of single cells while preserving network-level organization. We extend upon these results by constructing model networks constrained by experimental measurements and using them to probe the effect of biophysical parameters on network-level activity. We find in simulations that gamma-frequency oscillations are enabled by a high level of incoherent synaptic conductance input, similar to the barrage of noisy synaptic input that cortical neurons have been shown to receive in vivo. This incoherent synaptic input increases the emergent network frequency by shortening the time scale of the membrane in excitatory neurons and by reducing the temporal separation between excitation and inhibition due to decreased spike latency in inhibitory neurons. These mechanisms are demonstrated in simulations and in vitro current-clamp and dynamic-clamp experiments. Simulation results further indicate that the membrane potential noise amplitude has a large impact on network frequency and that the balance between excitatory and inhibitory currents controls network stability and sensitivity to external inputs. PMID:22275859

Timing of cord clamping in very preterm infants: more evidence is needed.

PubMed

Tarnow-Mordi, William O; Duley, Lelia; Field, David; Marlow, Neil; Morris, Jonathan; Newnham, John; Paneth, Nigel; Soll, Roger F; Sweet, David

2014-08-01

In December 2012, the American College of Obstetricians and Gynecologists published a Committee Opinion entitled "Timing of umbilical cord clamping after birth." It stated that "evidence exists to support delayed cord clamping in preterm infants, when feasible. The single most important benefit for preterm infants is the possibility for a nearly 50% reduction in IVH." However, the Committee Opinion added that the ideal timing of umbilical cord clamping has yet to be determined and recommended that large clinical trials be conducted in the most preterm infants. Published randomized controlled trials include <200 infants of <30 weeks' gestation, with assessments of neurodevelopmental outcome in less than one-half of the children. This is a major gap in the evidence. Without reliable data from randomized controlled trials that optimally include childhood follow-up evaluations, we will not know whether delayed cord clamping may do more overall harm than good. Ongoing trials of delayed cord clamping plan to report childhood outcomes in >2000 additional very preterm infants. Current recommendations may need to change when these results become available. Greater international collaboration could accelerate resolution of whether this promising intervention will improve disability-free survival in about 1 million infants who will be born very preterm globally each year. Copyright © 2014 Mosby, Inc. All rights reserved.

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.

Structural basis of drugs that increase cardiac inward rectifier Kir2.1 currents.

PubMed

Gómez, Ricardo; Caballero, Ricardo; Barana, Adriana; Amorós, Irene; De Palm, Sue-Haida; Matamoros, Marcos; Núñez, Mercedes; Pérez-Hernández, Marta; Iriepa, Isabel; Tamargo, Juan; Delpón, Eva

2014-11-01

We hypothesize that some drugs, besides flecainide, increase the inward rectifier current (IK1) generated by Kir2.1 homotetramers (IKir2.1) and thus, exhibit pro- and/or antiarrhythmic effects particularly at the ventricular level. To test this hypothesis, we analysed the effects of propafenone, atenolol, dronedarone, and timolol on Kir2.x channels. Currents were recorded with the patch-clamp technique using whole-cell, inside-out, and cell-attached configurations. Propafenone (0.1 nM-1 µM) did not modify either IK1 recorded in human right atrial myocytes or the current generated by homo- or heterotetramers of Kir2.2 and 2.3 channels recorded in transiently transfected Chinese hamster ovary cells. On the other hand, propafenone increased IKir2.1 (EC50 = 12.0 ± 3.0 nM) as a consequence of its interaction with Cys311, an effect which decreased inward rectification of the current. Propafenone significantly increased mean open time and opening frequency at all the voltages tested, resulting in a significant increase of the mean open probability of the channel. Timolol, which interacted with Cys311, was also able to increase IKir2.1. On the contrary, neither atenolol nor dronedarone modified IKir2.1. Molecular modelling of the Kir2.1-drugs interaction allowed identification of the pharmacophore of drugs that increase IKir2.1. Kir2.1 channels exhibit a binding site determined by Cys311 that is responsible for drug-induced IKir2.1 increase. Drug binding decreases channel affinity for polyamines and current rectification, and can be a mechanism of drug-induced pro- and antiarrhythmic effects not considered until now. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

Glucose concentrations modulate brain-derived neurotrophic factor responsiveness of neurones in the paraventricular nucleus of the hypothalamus.

PubMed

McIsaac, W; Ferguson, A V

2017-04-01

The hypothalamic paraventricular nucleus (PVN) is critical for normal energy balance and has been shown to contain high levels of both brain-derived neurotrophic factor (BDNF) and tropomyosin-receptor kinase B mRNA. Microinjections of BDNF into the PVN increase energy expenditure, suggesting that BDNF plays an important role in energy homeostasis through direct actions in this nucleus. The present study aimed to examine the postsynaptic effects of BDNF on the membrane potential of PVN neurones, and also to determine whether extracellular glucose concentrations modulated these effects. We used hypothalamic PVN slices from male Sprague-Dawley rats to perform whole cell current-clamp recordings from PVN neurones. BDNF was bath applied at a concentration of 2 nmol L -1 and the effects on membrane potential determined. BDNF caused depolarisations in 54% of neurones (n=25; mean±SEM, 8.9±1.2 mV) and hyperpolarisations in 23% (n=11; -6.7±1.4 mV), whereas the remaining cells were unaffected. These effects were maintained in the presence of tetrodotoxin (n=9; 56% depolarised, 22% hyperpolarised, 22% nonresponders), or the GABA a antagonist bicuculline (n=12; 42% depolarised, 17% hyperpolarised, 41% nonresponders), supporting the conclusion that these effects on membrane potential were postsynaptic. Current-clamp recordings from PVN neurones next examined the effects of BDNF on these neurones at varying extracellular glucose concentrations. Larger proportions of PVN neurones hyperpolarised in response to BDNF as the glucose concentrations decreased [10 mmol L -1 glucose 23% (n=11) of neurones hyperpolarised, whereas, at 0.2 mmol L -1 glucose, 71% showed hyperpolarising effects (n=12)]. Our findings reveal that BDNF has direct GABA A independent effects on PVN neurones, which are modulated by local glucose concentrations. The latter observation further emphasises the critical importance of using physiologically relevant conditions in an investigation of the central pathways involved in the regulation of energy homeostasis. © 2017 British Society for Neuroendocrinology.

Localization and function of the Kv3.1b subunit in the rat medulla oblongata: focus on the nucleus tractus solitarii

PubMed Central

Dallas, Mark L; Atkinson, Lucy; Milligan, Carol J; Morris, Neil P; Lewis, David I; Deuchars, Susan A; Deuchars, Jim

2005-01-01

The voltage-gated potassium channel subunit Kv3.1 confers fast firing characteristics to neurones. Kv3.1b subunit immunoreactivity (Kv3.1b-IR) was widespread throughout the medulla oblongata, with labelled neurones in the gracile, cuneate and spinal trigeminal nuclei. In the nucleus of the solitary tract (NTS), Kv3.1b-IR neurones were predominantly located close to the tractus solitarius (TS) and could be GABAergic or glutamatergic. Ultrastructurally, Kv3.1b-IR was detected in NTS terminals, some of which were vagal afferents. Whole-cell current-clamp recordings from neurones near the TS revealed electrophysiological characteristics consistent with the presence of Kv3.1b subunits: short duration action potentials (4.2 ± 1.4 ms) and high firing frequencies (68.9 ± 5.3 Hz), both sensitive to application of TEA (0.5 mm) and 4-aminopyridine (4-AP; 30 μm). Intracellular dialysis of an anti-Kv3.1b antibody mimicked and occluded the effects of TEA and 4-AP in NTS and dorsal column nuclei neurones, but not in dorsal vagal nucleus or cerebellar Purkinje cells (which express other Kv3 subunits, but not Kv3.1b). Voltage-clamp recordings from outside-out patches from NTS neurones revealed an outward K+ current with the basic characteristics of that carried by Kv3 channels. In NTS neurones, electrical stimulation of the TS evoked EPSPs and IPSPs, and TEA and 4-AP increased the average amplitude and decreased the paired pulse ratio, consistent with a presynaptic site of action. Synaptic inputs evoked by stimulation of a region lacking Kv3.1b-IR neurones were not affected, correlating the presence of Kv3.1b in the TS with the pharmacological effects. PMID:15528247

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.

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.

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.

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

Evidence that protons act as neurotransmitters at vestibular hair cell-calyx afferent synapses.

PubMed

Highstein, Stephen M; Holstein, Gay R; Mann, Mary Anne; Rabbitt, Richard D

2014-04-08

Present data support the conclusion that protons serve as an important neurotransmitter to convey excitatory stimuli from inner ear type I vestibular hair cells to postsynaptic calyx nerve terminals. Time-resolved pH imaging revealed stimulus-evoked extrusion of protons from hair cells and a subsequent buildup of [H(+)] within the confined chalice-shaped synaptic cleft (ΔpH ∼ -0.2). Whole-cell voltage-clamp recordings revealed a concomitant nonquantal excitatory postsynaptic current in the calyx terminal that was causally modulated by cleft acidification. The time course of [H(+)] buildup limits the speed of this intercellular signaling mechanism, but for tonic signals such as gravity, protonergic transmission offers a significant metabolic advantage over quantal excitatory postsynaptic currents--an advantage that may have driven the proliferation of postsynaptic calyx terminals in the inner ear vestibular organs of contemporary amniotes.

Safety evaluation of large external fixation clamps and frames in a magnetic resonance environment.

PubMed

Luechinger, Roger; Boesiger, Peter; Disegi, John A

2007-07-01

Large orthopedic external fixation clamps and related components were evaluated for force, torque, and heating response when subjected to the strong electromagnetic fields of magnetic-resonance (MR) imaging devices. Forces induced by a 3-Tesla (T) MR scanner were compiled for newly designed nonmagnetic clamps and older clamps that contained ferromagnetic components. Heating trials were performed in a 1.5 and in a 3 T MR scanner with two assembled external fixation frames. Forces of the newly designed clamps were more than a factor 2 lower as the gravitational force on the device whereas, magnetic forces on the older devices showed over 10 times the force induced by earth acceleration of gravity. No torque effects could be found for the newly designed clamps. Temperature measurements at the tips of Schanz screws in the 1.5 T MR scanner showed a rise of 0.7 degrees C for a pelvic frame and of 2.1 degrees C for a diamond knee bridge frame when normalized to a specific absorption rate (SAR) of 2 W/kg. The normalized temperature increases in the 3 T MR scanner were 0.9 degrees C for the pelvic frame and 1.1 degrees C for the knee bridge frame. Large external fixation frames assembled with the newly designed clamps (390 Series Clamps), carbon fiber reinforced rods, and implant quality 316L stainless steel Schanz screws met prevailing force and torque limits when tested in a 3-T field, and demonstrated temperature increase that met IEC-60601 guidelines for extremities. The influence of frame-induced eddy currents on the risk of peripheral nerve stimulation was not investigated. Copyright 2006 Wiley Periodicals, Inc.

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

Effects of pine needle extract on pacemaker currents in interstitial cells of Cajal from the murine small intestine.

PubMed

Cheong, Hyeonsook; Paudyal, Dilli Parasad; Jun, Jae Yeoul; Yeum, Cheol Ho; Yoon, Pyung Jin; Park, Chan Guk; Kim, Man Yoo; So, Insuk; Kim, Ki Whan; Choi, Seok

2005-10-31

Extracts of pine needles (Pinus densiflora Sieb. et Zucc.) have diverse physiological and pharmacological actions. In this study we show that pine needle extract alters pacemaker currents in interstitial cells of Cajal (ICC) by modulating ATP-sensitive K+ channels and that this effect is mediated by prostaglandins. In whole cell patches at 30 degrees , ICC generated spontaneous pacemaker potentials in the current clamp mode (I = 0), and inward currents (pacemaker currents) in the voltage clamp mode at a holding potential of -70 mV. Pine needle extract hyperpolarized the membrane potential, and in voltage clamp mode decreased both the frequency and amplitude of the pacemaker currents, and increased the resting currents in the outward direction. It also inhibited the pacemaker currents in a dose-dependent manner. Because the effects of pine needle extract on pacemaker currents were the same as those of pinacidil (an ATP-sensitive K+ channel opener) we tested the effect of glibenclamide (an ATP-sensitive K+ channels blocker) on ICC exposed to pine needle extract. The effects of pine needle extract on pacemaker currents were blocked by glibenclamide. To see whether production of prostaglandins (PGs) is involved in the inhibitory effect of pine needle extract on pacemaker currents, we tested the effects of naproxen, a non-selective cyclooxygenase (COX-1 and COX-2) inhibitor, and AH6809, a prostaglandin EP1 and EP2 receptor antagonist. Naproxen and AH6809 blocked the inhibitory effects of pine needle extract on ICC. These results indicate that pine needle extract inhibits the pacemaker currents of ICC by activating ATP-sensitive K+ channels via the production of PGs.

Open carbon nanopipettes as resistive-pulse sensors, rectification sensors, and electrochemical nanoprobes.

PubMed

Hu, Keke; Wang, Yixian; Cai, Huijing; Mirkin, Michael V; Gao, Yang; Friedman, Gary; Gogotsi, Yury

2014-09-16

Nanometer-sized glass and quartz pipettes have been widely used as a core of chemical sensors, patch clamps, and scanning probe microscope tips. Many of those applications require the control of the surface charge and chemical state of the inner pipette wall. Both objectives can be attained by coating the inner wall of a quartz pipette with a nanometer-thick layer of carbon. In this letter, we demonstrate the possibility of using open carbon nanopipettes (CNP) produced by chemical vapor deposition as resistive-pulse sensors, rectification sensors, and electrochemical nanoprobes. By applying a potential to the carbon layer, one can change the surface charge and electrical double-layer at the pipette wall, which, in turn, affect the ion current rectification and adsorption/desorption processes essential for resistive-pulse sensors. CNPs can also be used as versatile electrochemical probes such as asymmetric bipolar nanoelectrodes and dual electrodes based on simultaneous recording of the ion current through the pipette and the current produced by oxidation/reduction of molecules at the carbon nanoring.

Conductivity noise in transmembrane ion channels due to ion concentration fluctuations via diffusion.

PubMed

Mak, D O; Webb, W W

1997-03-01

A Green's function approach is developed from first principles to evaluate the power spectral density of conductance fluctuations caused by ion concentration fluctuations via diffusion in an electrolyte system. This is applied to simple geometric models of transmembrane ion channels to obtain an estimate of the magnitude of ion concentration fluctuation noise in the channel current. Pure polypeptide alamethicin forms stable ion channels with multiple conductance states in artificial phospholipid bilayers isolated onto tips of micropipettes with gigaohm seals. In the single-channel current recorded by voltage-clamp techniques, excess noise was found after the background instrumental noise and the intrinsic Johnson and shot noises were removed. The noise que to ion concentration fluctuations via diffusion was isolated by the dependence of the excess current noise on buffer ion concentration. The magnitude of the concentration fluctuation noise derived from experimental data lies within limits estimated using our simple geometric channel models. Variation of the noise magnitude for alamethicin channels in various conductance states agrees with theoretical prediction.

Different spatial distributions of sodium channels in the slowly and rapidly adapting stretch receptor neuron of the crayfish.

PubMed

Lin, J H; Rydqvist, B

1999-06-05

Inward Na+ currents were studied, using a two-microelectrode intracellular voltage-clamp technique, in the slowly adapting (SA) and rapidly adapting (RA) stretch receptor neurons of the crayfish after the axons were cut at different distances from the soma. In the SA neuron, inward Na+ currents were recorded in the soma even when the axon was cut as close as 100 microm from the center of the soma, indicating the presence of Na+ channels in these parts. Also, two populations of Na+ channels seem to exist in the SA neuron. In the RA neuron, only minute Na+ currents were observed if the axon was shorter than 250 microm. The results strongly indicate that the voltage-gated Na+ channels in the SA and RA neurons have different distributions and that the difference in the spatial distribution of Na+ channel types may be important for the difference in firing properties in the two types of neurons. Copyright 1999 Elsevier Science B.V.

Currents through Hv1 channels deplete protons in their vicinity.

PubMed

De-la-Rosa, Víctor; Suárez-Delgado, Esteban; Rangel-Yescas, Gisela E; Islas, León D

2016-02-01

Proton channels have evolved to provide a pH regulatory mechanism, affording the extrusion of protons from the cytoplasm at all membrane potentials. Previous evidence has suggested that channel-mediated acid extrusion could significantly change the local concentration of protons in the vicinity of the channel. In this work, we directly measure the proton depletion caused by activation of Hv1 proton channels using patch-clamp fluorometry recordings from channels labeled with the Venus fluorescent protein at intracellular domains. The fluorescence of the Venus protein is very sensitive to pH, thus behaving as a genetically encoded sensor of local pH. Eliciting outward proton currents increases the fluorescence intensity of Venus. This dequenching is related to the magnitude of the current and not to channel gating and is dependent on the pH gradient. Our results provide direct evidence of local proton depletion caused by flux through the proton-selective channel. © 2016 De-la-Rosa et al.

Ionic channel mechanisms mediating the intrinsic excitability of Kenyon cells in the mushroom body of the cricket brain.

PubMed

Inoue, Shigeki; Murata, Kaoru; Tanaka, Aiko; Kakuta, Eri; Tanemura, Saori; Hatakeyama, Shiori; Nakamura, Atsunao; Yamamoto, Chihiro; Hasebe, Masaharu; Kosakai, Kumiko; Yoshino, Masami

2014-09-01

Intrinsic neurons within the mushroom body of the insect brain, called Kenyon cells, play an important role in olfactory associative learning. In this study, we examined the ionic mechanisms mediating the intrinsic excitability of Kenyon cells in the cricket Gryllus bimaculatus. A perforated whole-cell clamp study using β-escin indicated the existence of several inward and outward currents. Three types of inward currents (INaf, INaP, and ICa) were identified. The transient sodium current (INaf) activated at -40 mV, peaked at -26 mV, and half-inactivated at -46.7 mV. The persistent sodium current (INaP) activated at -51 mV, peaked at -23 mV, and half-inactivated at -30.7 mV. Tetrodotoxin (TTX; 1 μM) completely blocked both INaf and INaP, but 10nM TTX blocked INaf more potently than INaP. Cd(2+) (50 μM) potently blocked INaP with little effect on INaf. Riluzole (>20 μM) nonselectively blocked both INaP and INaf. The voltage-dependent calcium current (ICa) activated at -30 mV, peaked at -11.3 mV, and half-inactivated at -34 mV. The Ca(2+) channel blocker verapamil (100 μM) blocked ICa in a use-dependent manner. Cell-attached patch-clamp recordings showed the presence of a large-conductance Ca(2+)-activated K(+) (BK) channel, and the activity of this channel was decreased by removing the extracellular Ca(2+) or adding verapamil or nifedipine, and increased by adding the Ca(2+) agonist Bay K8644, indicating that Ca(2+) entry via the L-type Ca(2+) channel regulates BK channel activity. Under the current-clamp condition, membrane depolarization generated membrane oscillations in the presence of 10nM TTX or 100 μM riluzole in the bath solution. These membrane oscillations disappeared with 1 μM TTX, 50 μM Cd(2+), replacement of external Na(+) with choline, and blockage of Na(+)-activated K(+) current (IKNa) with 50 μM quinidine, indicating that membrane oscillations are primarily mediated by INaP in cooperation with IKNa. The plateau potentials observed either in Ca(2+)-free medium or in the presence of verapamil were eliminated by blocking INaP with 50 μM Cd(2+). Taken together, these results indicate that INaP and IKNa participate in the generation of membrane oscillations and that INaP additionally participates in the generation of plateau potentials and initiation of spontaneous action potentials. ICa, through L-type Ca(2+) channels, was also found to play a role in the rapid membrane repolarization of action potentials by functional coupling with BK channels. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

Two distinct classes of functional α7-containing nicotinic receptor on rat superior cervical ganglion neurons

PubMed Central

Cuevas, Javier; Roth, Adelheid L; Berg, Darwin K

2000-01-01

Nicotinic acetylcholine receptors (nAChRs) that bind α-bungarotoxin (αBgt) were studied on isolated rat superior cervical ganglion (SCG) neurons using whole-cell patch clamp recording techniques.Rapid application of ACh onto the soma of voltage clamped neurons evoked a slowly desensitizing current that was reversibly blocked by αBgt (50 nm). The toxin-sensitive current constituted on average about half of the peak whole-cell response evoked by ACh.Nanomolar concentrations of methyllycaconitine blocked the αBgt-sensitive component of the ACh-evoked current as did intracellular dialysis with an anti-α7 monoclonal antibody. The results indicate that the slowly reversible toxin-sensitive response elicited by ACh arises from activation of an unusual class of α7-containing receptor (α7-nAChR) similar to that reported previously for rat intracardiac ganglion neurons.A second class of functional α7-nAChR was identified on some SCG neurons by using rapid application of choline to elicit responses. In these cases a biphasic response was obtained, which included a rapidly desensitizing component that was blocked by αBgt in a pseudo-irreversible manner. The pharmacology and kinetics of the responses resembled those previously attributed to α7-nAChRs in a number of other neuronal cell types.Experiments measuring the dissociation rate of 125I-labelled αBgt from SCG neurons revealed two classes of toxin-binding site. The times for toxin dissociation were consistent with those required to reverse blockade of the two kinds of αBgt-sensitive response.These results indicate that rat SCG neurons express two types of functional α7-nAChR, differing in pharmacology, desensitization and reversibility of αBgt blockade. PMID:10856125

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.

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

Systemic administration of anti-NGF increases A-type potassium currents and decreases pancreatic nociceptor excitability in a rat model of chronic pancreatitis.

PubMed

Zhu, Yaohui; Mehta, Kshama; Li, Cuiping; Xu, Guang-Yin; Liu, Liansheng; Colak, Tugba; Shenoy, Mohan; Pasricha, Pankaj Jay

2012-01-01

We have previously shown that pancreatic sensory neurons in rats with chronic pancreatitis (CP) display increased excitability associated with a decrease in transient inactivating potassium currents (I(A)), thus accounting in part for the hyperalgesia associated with this condition. Because of its well known role in somatic hyperalgesia, we hypothesized a role for the nerve growth factor (NGF) in driving these changes. CP was induced by intraductal injection of trinitrobenzene sulfonic acid (TNBS) in rats. After 3 wk, anti-NGF antibody or control serum was injected intra-peritoneally daily for 1 wk. This protocol was repeated in another set of experiments in control rats (receiving intraductal PBS instead of TNBS). Pancreatic nociceptors labeled with the dye Dil were identified, and patch-clamp recordings were made from acutely dissociated DRG neurons. Sensory neurons from anti-NGF-treated rats displayed a lower resting membrane potential, increased rheobase, decreased burst discharges in response to stimulatory current, and decreased input resistance compared with those treated with control serum. Under voltage-clamp condition, neuronal I(A) density was increased in anti-NGF-treated rats compared with rats treated with control serum. However, anti-NGF treatment had no effect on electrophysiological parameters in neurons from control rats. The expression of Kv-associated channel or ancillary genes Kv1.4, 4.1, 4.2, 4.3, and DPP6, DPP10, and KCHIPs 1-4 in pancreas-specific nociceptors was examined by laser-capture microdissection and real-time PCR quantification of mRNA levels. No significant differences were seen among those. These findings emphasize a key role for NGF in maintaining neuronal excitability in CP specifically via downregulation of I(A) by as yet unknown mechanisms.

Potential Danger of Pre-Pump Clamping on Negative Pressure-Associated Gaseous Microemboli Generation During Extracorporeal Life Support--An In Vitro Study.

PubMed

Wang, Shigang; Chin, Brian J; Gentile, Frank; Kunselman, Allen R; Palanzo, David; Ündar, Akif

2016-01-01

The objectives of this study were to investigate the relationship between revolution speed of a conventional centrifugal pump and negative pressure at the inlet of the pump by clamping the tubing upstream of the pump, and to verify whether negative pressure leads to gaseous microemboli (GME) production in a simulated adult extracorporeal life support (ECLS) system. The experimental circuit, including a Maquet Rotaflow centrifugal pump and a Medos Hilite 7000 LT polymethyl-pentene membrane oxygenator, was primed with packed red blood cells (hematocrit 35%). Negative pressure was created in the circuit by clamping the tubing upstream of the pump for 10 s, and then releasing the clamp. An emboli detection and classification quantifier was used to record GME volume and count at pre-oxygenator and post-oxygenator sites, and pressure and flow rate data were collected using a custom-based data acquisition system. All trials were conducted at 36°C at revolution speeds of 2000-4000 rpm (500 rpm increment). The flow rates were 1092.5-4708.4 mL/min at the revolution speeds of 2000-4000 rpm. Higher revolution speed generated higher negative pressure at the pre-pump site when clamping the tubing upstream of the pump (-108.3 ± 0.1 to -462.0 ± 0.5 mm Hg at 2000-4000 rpm). Moreover, higher negative pressure was associated with a larger number and volume of GME at pre-oxygenator site after de-clamp (GME count 10,573 ± 271 at pre-oxygenator site at 4000 rpm). The results showed that there was a potential danger of delivering GME to the patient when clamping pre-pump tubing during ECLS using a centrifugal pump. Our results warrant further clinical studies to investigate this phenomenon. Copyright © 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Impact of delayed umbilical cord clamping on public cord blood donations: can we help future patients and benefit infant donors?

PubMed

Ciubotariu, Rodica; Scaradavou, Andromachi; Ciubotariu, Ilinca; Tarnawski, Michal; Lloyd, Sara; Albano, Maria; Dobrila, Ludy; Rubinstein, Pablo; Grunebaum, Amos

2018-03-25

Cord blood (CB) is a widely accepted stem cell source and its clinical utilization depends, to a great extent, on its cell content. Birth-to-clamping (BTC) time of umbilical cord determines placental transfusion to the newborn, and the remaining blood that can be collected and banked. The 2017 Committee Opinion of the American College of Obstetrics and Gynecologists (ACOG) recommends a delay of "at least 30-60 seconds" before clamping the cord for all newborns to ensure adequate iron stores. The impact of delayed cord clamping (DCC) on public CB banking can be substantial. Cord blood units (CBUs) collected from 1210 mothers at one hospital were evaluated for total nucleated cells (TNCs) and weight/volume based on time to clamping. Bank staff recorded BTC time in seconds as reported by obstetricians; collections were performed ex utero. Immediate clamping was defined as BTC of less than 30 seconds, whereas DCC was defined as BTC of 30 seconds or more. Cord clamping was immediate in 903 (75%) and delayed in 307 (25%) deliveries. Successful recovery (% clinical CBUs) decreased 10-fold with DCC of more than 60 seconds (22% vs. 2.4%, p < 0.001). CBUs collected after DCC of more than 60 seconds had significantly lower TNC counts than those after DCC of less than 60 seconds (p < 0.0001). Furthermore, 38% to 46% of CBUs after DCC of more than 60 seconds had volume of less than 40 mL. Our study indicates that DCC of 30 to 60 seconds has a small negative impact on collection of high-TNC-count CBUs. However, increasing BTC to more than 60 seconds decreases significantly both TNC content and volume, reducing drastically the chances of obtaining clinically useful CBUs. © 2018 AABB.

Direct demonstration of persistent Na+ channel activity in dendritic processes of mammalian cortical neurones

PubMed Central

Magistretti, Jacopo; Ragsdale, David S; Alonso, Angel

1999-01-01

Single Na+ channel activity was recorded in patch-clamp, cell-attached experiments performed on dendritic processes of acutely isolated principal neurones from rat entorhinal-cortex layer II. The distances of the recording sites from the soma ranged from ≈20 to ≈100 μm.Step depolarisations from holding potentials of −120 to −100 mV to test potentials of −60 to +10 mV elicited Na+ channel openings in all of the recorded patches (n= 16).In 10 patches, besides transient Na+ channel openings clustered within the first few milliseconds of the depolarising pulses, prolonged and/or late Na+ channel openings were also regularly observed. This ‘persistent’ Na+ channel activity produced net inward, persistent currents in ensemble-average traces, and remained stable over the entire duration of the experiments (≈9 to 30 min).Two of these patches contained <= 3 channels. In these cases, persistent Na+ channel openings could be attributed to the activity of one single channel.The voltage dependence of persistent-current amplitude in ensemble-average traces closely resembled that of whole-cell, persistent Na+ current expressed by the same neurones, and displayed the same characteristic low threshold of activation.Dendritic, persistent Na+ channel openings had relatively high single-channel conductance (≈20 pS), similar to what is observed for somatic, persistent Na+ channels.We conclude that a stable, persistent Na+ channel activity is expressed by proximal dendrites of entorhinal-cortex layer II principal neurones, and can contribute a significant low-threshold, persistent Na+ current to the dendritic processing of excitatory synaptic inputs. PMID:10601494

Effects of Xinjining extract on inward rectifier potassium current in ventricular myocytes of guinea pig.

PubMed

Zhu, Ming-jun; Wang, Guo-juan; Wang, Yong-xia; Pu, Jie-lin; Liu, Hong-jun; Yu, Hai-bin

2010-02-01

To study the effect of Xinjining extract (, XJN) on inward rectifier potassium current (I(K1)) in ventricular myocyte (VMC) of guinea pigs and its anti-arrhythmic mechanism on ion channel level. Single VMC was enzymatically isolated by zymolisis, and whole-cell patch clamp recording technique was used to record the I(k1) in VMC irrigated with XJN of different concentrations (1.25, 2.50, 5.00 g/L; six samples for each). The stable current and conductance of the inward component of I(K1) as well as the outward component of peak I(K1) and conductance of it accordingly was recorded when the test voltage was set on -110 mV. The suppressive rate of XJN on the inward component of I(K1) was 9.54% + or - 5.81%, 34.82% + or - 15.03%, and 59.52% + or - 25.58% with a concentration of 1.25, 2.50, and 5.00 g/L, respectively, and that for the outward component of peak I(K1) was 23.94% + or - 7.45%, 52.98% + or - 19.62%, and 71.42% + or - 23.01%, respectively (all P<0.05). Moreover, different concentrations of XJN also showed effects for reducing I(K1) conductance. XJN has inhibitory effect on I(K1) in guinea pig's VMC, and that of the same concentration shows stronger inhibition on outward component than on inward component, which may be one of the mechanisms of its anti-arrhythmic effect.

Characterization of L-type calcium channel activity in atrioventricular nodal myocytes from rats with streptozotocin-induced Diabetes mellitus

PubMed Central

Yuill, Kathryn H; Al Kury, Lina T; Howarth, Frank Christopher

2015-01-01

Cardiovascular complications are common in patients with Diabetes mellitus (DM). In addition to changes in cardiac muscle inotropy, electrical abnormalities are also commonly observed in these patients. We have previously shown that spontaneous cellular electrical activity is altered in atrioventricular nodal (AVN) myocytes, isolated from the streptozotocin (STZ) rat model of type-1 DM. In this study, utilizing the same model, we have characterized the changes in L-type calcium channel activity in single AVN myocytes. Ionic currents were recorded from AVN myocytes isolated from the hearts of control rats and from those with STZ-induced diabetes. Patch-clamp recordings were used to assess the changes in cellular electrical activity in individual myocytes. Type-1 DM significantly altered the cellular characteristics of L-type calcium current. A reduction in peak ICaL density was observed, with no corresponding changes in the activation parameters of the current. L-type calcium channel current also exhibited faster time-dependent inactivation in AVN myocytes from diabetic rats. A negative shift in the voltage dependence of inactivation was also evident, and a slowing of restitution parameters. These findings demonstrate that experimentally induced type-1 DM significantly alters AVN L-type calcium channel cellular electrophysiology. These changes in ion channel activity may contribute to the abnormalities in cardiac electrical function that are associated with high mortality levels in patients with DM. PMID:26603460

Current sensor

DOEpatents

Yakymyshyn, Christopher Paul; Brubaker, Michael Allen; Yakymyshyn, Pamela Jane

2007-01-16

A current sensor is described that uses a plurality of magnetic field sensors positioned around a current carrying conductor. The sensor can be hinged to allow clamping to a conductor. The current sensor provides high measurement accuracy for both DC and AC currents, and is substantially immune to the effects of temperature, conductor position, nearby current carrying conductors and aging.

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

Characteristics of action potentials and their underlying outward currents in rat taste receptor cells.

PubMed

Chen, Y; Sun, X D; Herness, S

1996-02-01

1. Taste receptor cells produce action potentials as a result of transduction mechanisms that occur when these cells are stimulated with tastants. These action potentials are thought to be key signaling events in relaying information to the central nervous system. We explored the ionic basis of action potentials from dissociated posterior rat taste cells using the patch-clamp recording technique in both voltage-clamp and current-clamp modes. 2. Action potentials were evoked by intracellular injection of depolarizing current pulses from a holding potential of -80 mV. The threshold potential for firing of action potentials was approximately -35 mV; the input resistance of these cells averaged 6.9 G omega. With long depolarizing pulses, two or three action potentials could be elicited with successive attenuation of the spike height. Afterhyperpolarizations were observed often. 3. Both sodium and calcium currents contribute to depolarizing phases of the action potential. Action potentials were blocked completely in the presence of the sodium channel blocker tetrodotoxin. Calcium contributions could be visualized as prolonged calcium plateaus when repolarizing potassium currents were blocked and barium was used as a charge carrier. 4. Outward currents were composed of sustained delayed rectifier current, transient potassium current, and calcium-activated potassium current. Transient and sustained potassium currents activated close to -30 mV and increased monotonically with further depolarization. Up to half the outward current inactivated with decay constants on the order of seconds. Sustained and transient currents displayed steep voltage dependence in conductance and inactivation curves. Half inactivation occurred at -20 +/- 3.1 mV (mean +/- SE) with a decrease of 11.2 +/- 0.5 mV per e-fold. Half maximal conductance occurred at 3.6 +/- 1.8 mV and increased 12.2 +/- 0.6 mV per e-fold. Calcium-activated potassium current was evidenced by application of apamin and the use of calcium-free bathing solution. It was most obvious at more depolarized holding potentials that inactivated much of the transient and sustained outward currents. 5. Potassium currents contribute to both the repolarization and afterhyperpolarization phases of the action potential. These currents were blocked by bath application of tetraethylammonium, which also substantially broadened the action potential. Application of 4-aminopyridine was able to selectively block transient potassium currents without affecting sustained currents. This also broadened the action potential as well as eliminated the afterhyperpolarization. 6. A second type of action potential was observed that differed in duration. These slow action potentials had t1/2 durations of 9.6 ms compared with 1.4 ms for fast action potentials. Input resistances of the two groups were indistinguishable. Approximately one-fourth of the cells eliciting action potentials were of the slow type. 7. Cells eliciting fast action potentials had large outward currents capable of producing a quick repolarization, whereas cells with slow action potentials had small outward currents by comparison. The average values of fast cells were 2,563 pA and 1.4 ms compared with 373 pA and 9.6 ms for slow cells. Current and duration values were related exponentially. No significant difference was noted for inward currents. 8. These results suggest that many taste receptor cells conduct action potentials, which may be classified broadly into two groups on the basis of action potential duration and potassium current magnitude. These groups may be related to cell turnover. The physiological role of action potentials remains to be elucidated but may be important for communication within the taste bud as well as to the afferent nerve.

Enhanced functional expression of transient outward current in hypertrophied feline myocytes.

PubMed

Ten Eick, R E; Zhang, K; Harvey, R D; Bassett, A L

1993-08-01

Cardiac hypertrophy can decrease myocardial contractility and alter the electrophysiological activity of the heart. It is well documented that action potentials recorded from hypertrophied feline ventricular cells can exhibit depressed plateau voltages and prolonged durations. Similar findings have been made by others in rabbit, rat, guinea pig, and human heart. Whole-cell patch voltage-clamp studies designed to explain these changes in the action potential suggest that the only component of the membrane current recorded from feline right ventricular (RV) myocytes found to be substantially different from normal is the 4-amino-pyridine-sensitive transient outward current (I(to)). However, it was not clear if the change in I(to) could explain the changes in the action potential of hypertrophied cardiocytes, nor was it clear if these changes reflect an alteration in the electrophysiological character of the channels underlying I(to). A kinetic comparison of I(to) elicited by hypertrophied RV myocytes with that elicited by comparable normal RV myocytes previously revealed no differences, suggesting that the increased magnitude of the peak I(to) recorded from hypertrophied myocytes arises because the current density increases and not because of any alteration in the kinetic parameters governing the current. This finding suggests that in hypertrophy additional normal channels are expressed rather than a kinetically different channel subtype emerging. Investigations designed to determine if enhancement of I(to) could explain the hypertrophy-induced changes in plateau voltage and action potential duration suggest that a change in I(to) density can indeed explain the entire effect of hypertrophy on RV action potentials. If this notion is correct, the likelihood of "sudden death" in patients with myocardial hypertrophy might be decreased by a blocker selective for cardiac I(to).

An ALS-Associated Mutant SOD1 Rapidly Suppresses KCNT1 (Slack) Na+-Activated K+ Channels in Aplysia Neurons.

PubMed

Zhang, Yalan; Ni, Weiming; Horwich, Arthur L; Kaczmarek, Leonard K

2017-02-22

Mutations that alter levels of Slack (KCNT1) Na + -activated K + current produce devastating effects on neuronal development and neuronal function. We now find that Slack currents are rapidly suppressed by oligomers of mutant human Cu/Zn superoxide dismutase 1 (SOD1), which are associated with motor neuron toxicity in an inherited form of amyotrophic lateral sclerosis (ALS). We recorded from bag cell neurons of Aplysia californica , a model system to study neuronal excitability. We found that injection of fluorescent wild-type SOD1 (wt SOD1YFP) or monomeric mutant G85R SOD1YFP had no effect on net ionic currents measured under voltage clamp. In contrast, outward potassium currents were significantly reduced by microinjection of mutant G85R SOD1YFP that had been preincubated at 37°C or of cross-linked dimers of G85R SOD1YFP. Reduction of potassium current was also seen with multimeric G85R SOD1YFP of ∼300 kDa or >300 kDa that had been cross-linked. In current clamp recordings, microinjection of cross-linked 300 kDa increased excitability by depolarizing the resting membrane potential, and decreasing the latency of action potentials triggered by depolarization. The effect of cross-linked 300 kDa on potassium current was reduced by removing Na + from the bath solution, or by knocking down levels of Slack using siRNA. It was also prevented by pharmacological inhibition of ASK1 (apoptosis signal-regulating kinase 1) or of c-Jun N-terminal kinase, but not by an inhibitor of p38 mitogen-activated protein kinase. These results suggest that soluble mutant SOD1 oligomers rapidly trigger a kinase pathway that regulates the activity of Na + -activated K + channels in neurons. SIGNIFICANCE STATEMENT Slack Na + -activated K + channels (KCNT1, K Na 1.1) regulate neuronal excitability but are also linked to cytoplasmic signaling pathways that control neuronal protein translation. Mutations that alter the amplitude of these currents have devastating effects on neuronal development and function. We find that injection of oligomers of mutant superoxide dismutase 1 (SOD1) into the cytoplasm of invertebrate neurons rapidly suppresses these Na + -activated K + currents and that this effect is mediated by a MAP kinase cascade, including ASK1 and c-Jun N-terminal kinase. Because amyotrophic lateral sclerosis is a fatal adult-onset neurodegenerative disease produced by mutations in SOD1 that cause the enzyme to form toxic oligomers, our findings suggest that suppression of Slack channels may be an early step in the progression of the disease. Copyright © 2017 the authors 0270-6474/17/372258-08$15.00/0.

Nicotinic excitation of rat ventral tegmental neurones in vitro studied by intracellular recording.

PubMed Central

Calabresi, P.; Lacey, M. G.; North, R. A.

1989-01-01

1. Intracellular recordings were made from presumed dopamine-containing neurones in the ventral tegmental area (VTA) in rat brain slices. 2. Nicotine (10-100 microM) and acetylcholine (ACh) depolarized the neurones. The depolarization caused by ACh was typically biphasic; both components were increased by neostigmine (0.1-10 microM), but only the slower component was blocked by scopolamine (1-10 microM). 3. The nicotinic action of ACh, studied in the presence of neostigmine and scopolamine, persisted in the presence of tetrodotoxin (1 microM) and cobalt (2-5 mM). 4. ACh or carbachol (30 microM) caused inward currents in neurones voltage-clamped near the resting potential. These currents reversed polarity at around -4 mV, were blocked by hexamethonium (1-100 microM) in a voltage-dependent manner, and showed desensitization with prolonged or repeated agonist applications. 5. Depolarizations caused by ACh and carbachol were reduced in slices pretreated with kappa-bungarotoxin, but were not changed by alpha-bungarotoxin. 6. These responses to ACh and nicotine resemble those previously described on autonomic ganglion cells. The direct action on VTA neurones may contribute to the positive reinforcement associated with nicotine consumption. PMID:2804543

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.

Hemodynamics on abrupt stoppage of centrifugal pumps during left ventricular assist.

PubMed

Kono, S; Nishimura, K; Nishina, T; Akamatsu, T; Komeda, M

2000-01-01

A magnetically suspended centrifugal pump (MSCP), developed for long-term ventricular assist, is reliable and durable because it has no shaft or seal. However, with nonvalve pumps such as a MSCP, regurgitation occurs when they accidentally stop without cannula clamping. We investigated the hemodynamics during temporary stoppage of a MSCP being used as a left ventricular assist system (LVAS), comparing two inflow cannulation sites. In four sheep (weight, 35-45 kg), microspheres were injected into the left main coronary artery to induce heart failure. An outflow cannula was sutured onto the descending aorta, and two inflow cannulae were inserted into the left atrium and the left ventricle. The MSCP was stopped with both the left ventricular cannula and left atrial cannula clamped, and the hemodynamics and P-V loops were recorded. Each cannula was then unclamped in order, and similar parameters were recorded. LVEDP increased at unclamping of the left ventricular cannula (ULVC), and rose further at unclamping of the left atrial cannula (ULAC). Aortic pressure did not change at ULVC, but decreased at ULAC. The effective systemic flow that subtracted the regurgitant flow through the MSCP from left ventricular output was half at ULVC and almost 0 at ULAC. When stopping centrifugal pumps without circuit clamping, hemodynamic deterioration is less at ULVC than at ULAC. This finding suggests that left ventricular inflow cannulation is recommended to allow more time in emergency situations.

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

High sensitivity of spontaneous spike frequency to sodium leak current in a Lymnaea pacemaker neuron.

PubMed

Lu, T Z; Kostelecki, W; Sun, C L F; Dong, N; Pérez Velázquez, J L; Feng, Z-P

2016-12-01

The spontaneous rhythmic firing of action potentials in pacemaker neurons depends on the biophysical properties of voltage-gated ion channels and background leak currents. The background leak current includes a large K + and a small Na + component. We previously reported that a Na + -leak current via U-type channels is required to generate spontaneous action potential firing in the identified respiratory pacemaker neuron, RPeD1, in the freshwater pond snail Lymnaea stagnalis. We further investigated the functional significance of the background Na + current in rhythmic spiking of RPeD1 neurons. Whole-cell patch-clamp recording and computational modeling approaches were carried out in isolated RPeD1 neurons. The whole-cell current of the major ion channel components in RPeD1 neurons were characterized, and a conductance-based computational model of the rhythmic pacemaker activity was simulated with the experimental measurements. We found that the spiking rate is more sensitive to changes in the Na + leak current as compared to the K + leak current, suggesting a robust function of Na + leak current in regulating spontaneous neuronal firing activity. Our study provides new insight into our current understanding of the role of Na + leak current in intrinsic properties of pacemaker neurons. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Ion channel recordings on an injection-molded polymer chip.

PubMed

Tanzi, Simone; Matteucci, Marco; Christiansen, Thomas Lehrmann; Friis, Søren; Christensen, Mette Thylstrup; Garnaes, Joergen; Wilson, Sandra; Kutchinsky, Jonatan; Taboryski, Rafael

2013-12-21

In this paper, we demonstrate recordings of the ion channel activity across the cell membrane in a biological cell by employing the so-called patch clamping technique on an injection-molded polymer microfluidic device. The findings will allow direct recordings of ion channel activity to be made using the cheapest materials and production platform to date and with the potential for very high throughput. The employment of cornered apertures for cell capture allowed the fabrication of devices without through holes and via a scheme comprising master origination by dry etching in a silicon substrate, electroplating in nickel and injection molding of the final part. The most critical device parameters were identified as the length of the patching capillary and the very low surface roughness on the inside of the capillary. The cross-sectional shape of the orifice was found to be less critical, as both rectangular and semicircular profiles seemed to have almost the same ability to form tight seals with cells with negligible leak currents. The devices were functionally tested using human embryonic kidney cells expressing voltage-gated sodium channels (Nav1.7) and benchmarked against a commercial state-of-the-art system for automated ion channel recordings. These experiments considered current-voltage (IV) relationships for activation and inactivation of the Nav1.7 channels and their sensitivity to a local anesthetic, lidocaine. Both IVs and lidocaine dose-response curves obtained from the injection-molded polymer device were in good agreement with data obtained from the commercial system.

Excitatory synaptic inputs to mouse on-off direction-selective retinal ganglion cells lack direction tuning.

PubMed

Park, Silvia J H; Kim, In-Jung; Looger, Loren L; Demb, Jonathan B; Borghuis, Bart G

2014-03-12

Direction selectivity represents a fundamental visual computation. In mammalian retina, On-Off direction-selective ganglion cells (DSGCs) respond strongly to motion in a preferred direction and weakly to motion in the opposite, null direction. Electrical recordings suggested three direction-selective (DS) synaptic mechanisms: DS GABA release during null-direction motion from starburst amacrine cells (SACs) and DS acetylcholine and glutamate release during preferred direction motion from SACs and bipolar cells. However, evidence for DS acetylcholine and glutamate release has been inconsistent and at least one bipolar cell type that contacts another DSGC (On-type) lacks DS release. Here, whole-cell recordings in mouse retina showed that cholinergic input to On-Off DSGCs lacked DS, whereas the remaining (glutamatergic) input showed apparent DS. Fluorescence measurements with the glutamate biosensor intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) conditionally expressed in On-Off DSGCs showed that glutamate release in both On- and Off-layer dendrites lacked DS, whereas simultaneously recorded excitatory currents showed apparent DS. With GABA-A receptors blocked, both iGluSnFR signals and excitatory currents lacked DS. Our measurements rule out DS release from bipolar cells onto On-Off DSGCs and support a theoretical model suggesting that apparent DS excitation in voltage-clamp recordings results from inadequate voltage control of DSGC dendrites during null-direction inhibition. SAC GABA release is the apparent sole source of DS input onto On-Off DSGCs.

Functional and structural effects of amyloid-β aggregate on Xenopus laevis oocytes.

PubMed

Parodi, Jorge; Ochoa-de la Paz, Lenin; Miledi, Ricardo; Martínez-Torres, Ataúlfo

2012-10-01

Xenopus laevis oocytes exposed to amyloid-β aggregate generated oscillatory electric activity (blips) that was recorded by two-microelectrode voltage-clamp. The cells exhibited a series of "spontaneous" blips ranging in amplitude from 3.8 ± 0.9 nA at the beginning of the recordings to 6.8 ± 1.7 nA after 15 min of exposure to 1 μM aggregate. These blips were similar in amplitude to those induced by the channel-forming antimicrobial agents amphotericin B (7.8 ± 1.2 nA) and gramicidin (6.3 ± 1.1 nA). The amyloid aggregate-induced currents were abolished when extracellular Ca(2+) was removed from the bathing solution, suggesting a central role for this cation in generating the spontaneous electric activity. The amyloid aggregate also affected the Ca(2+)-dependent Cl(-) currents of oocytes, as shown by increased amplitude of the transient-outward chloride current (T(out)) and the serum-activated, oscillatory Cl(-) currents. Electron microcopy revealed that amyloid aggregate induced the dissociation of the follicular cells that surround the oocyte, thus leading to a failure in the electro-chemical communication between these cells. This was also evidenced by the suppression of the oscillatory Ca(2+)-dependent ATP-currents, which require proper coupling between oocytes and the follicular cell layer. These observations, made using the X. laevis oocytes as a versatile experimental model, may help to understand the effects of amyloid aggregate on cellular communication.

PKC regulates capsaicin-induced currents of dorsal root ganglion neurons in rats.

PubMed

Zhou, Y; Zhou, Z S; Zhao, Z Q

2001-10-01

Capsaicin activates a non-specific cation conductance in a subset of dorsal root ganglion (DRG) neurons. The inward current and membrane potential of acutely isolated DRG neurons were examined using whole-cell patch recording methods. We report here that the current and voltage responses activated by capsaicin were markedly increased by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC). The mean current, after application of 0.3 microM PMA, was 153.5+/-5.7% of control (n=32) in Ca(2+)-free external solution and 181.6+/-6.8% of control (n=15) in standard external solution. Under current-clamp conditions, 0.3 microM PMA facilitated capsaicin-induced depolarization and action potential generation. Bindolylmaleimide I (BIM), a specific inhibitor of PKC activity, abolished the effect of PMA. In addition, capsaicin-evoked current was attenuated to 68.3+/-5.0% of control (n=13) by individual administration of 1 microM BIM in standard external solution, while 0.3 microM BIM did not have this effect. These data suggest that PKC can directly regulate the capsaicin response in DRG neurons, which could increase nociceptive sensory transmission and contribute to hyperalgesia.

A decay of gap junctions associated with ganglion cell differentiation during retinal regeneration of the adult newt.

PubMed

Oi, Hanako; Chiba, Chikafumi; Saito, Takehiko

2003-12-01

Changes in the gap junctional coupling and maturation of voltage-activated Na(+) currents during regeneration of newt retinas were examined by whole-cell patch-clamping in slice preparations. Progenitor cells in regenerating retinas did not exhibit Na(+) currents but showed prominent electrical and tracer couplings. Cells identified by LY-fills were typically slender. Na(+) currents were detected in premature ganglion cells with round somata in the 'intermediate-II' regenerating retina. No electrical and tracer couplings were observed between these cells. Mature ganglion cells did not exhibit electrical coupling, but showed tracer coupling. On average, the maximum Na(+) current amplitude recorded from premature ganglion cells was roughly 2.5-fold smaller than that of mature ganglion cells. In addition, the activation threshold of the Na(+) current was nearly 11 mV more positive than that of mature cells. We provide morphological and physiological evidence showing that loss of gap junctions between progenitor cells is associated with ganglion cell differentiation during retinal regeneration and that new gap junctions are recreated between mature ganglion cells. Also we provide evidence suggesting that the loss of gap junctions correlates with the appearance of voltage-activated Na(+) currents in ganglion cells.

Inhibitory effects of antihistamines, diphenhydramine and chlorpheniramine, on proton currents in BV2 microglial cells.

PubMed

Kim, Jiwon; Song, Jin-Ho

2017-03-05

Microglial NADPH oxidase is a major source of toxic reactive oxygen species produced during chronic neuroinflammation. Voltage-gated proton channel (H V 1) functions to maintain the intense activity of NADPH oxidase, and channel inhibition alleviates the pathology of neurodegenerative diseases such as ischemic stroke and multiple sclerosis associated with oxidative neuroinflammation. Antagonists of histamine H 1 receptors have beneficial effects against microglia-mediated oxidative stress and neurotoxicity. We examined the effects of the H 1 antihistamines, diphenhydramine and chlorpheniramine, on proton currents in BV2 microglial cells recorded using the whole-cell patch clamp technique. Diphenhydramine and chlorpheniramine reduced the proton currents with almost the same potency, yielding IC 50 values of 42 and 43μM, respectively. Histamine did not affect proton currents, excluding the involvement of histamine receptors in their action. Neither drug shifted the voltage-dependence of activation or the reversal potential of the proton currents, even though diphenhydramine slowed the activation and deactivation kinetics. The inhibitory effects of the two antihistamines on proton currents could be utilized to develop therapeutic agents for neurodegenerative diseases and other diseases associated with H V 1 proton channel abnormalities. Copyright © 2017 Elsevier B.V. All rights reserved.

Fault-tolerant three-level inverter

DOEpatents

Edwards, John; Xu, Longya; Bhargava, Brij B.

2006-12-05

A method for driving a neutral point clamped three-level inverter is provided. In one exemplary embodiment, DC current is received at a neutral point-clamped three-level inverter. The inverter has a plurality of nodes including first, second and third output nodes. The inverter also has a plurality of switches. Faults are checked for in the inverter and predetermined switches are automatically activated responsive to a detected fault such that three-phase electrical power is provided at the output nodes.

Activation of Mechanosensitive Transient Receptor Potential/Piezo Channels in Odontoblasts Generates Action Potentials in Cocultured Isolectin B4-negative Medium-sized Trigeminal Ganglion Neurons.

PubMed

Sato, Masaki; Ogura, Kazuhiro; Kimura, Maki; Nishi, Koichi; Ando, Masayuki; Tazaki, Masakazu; Shibukawa, Yoshiyuki

2018-06-01

Various stimuli to the dentin surface elicit dentinal pain by inducing dentinal fluid movement causing cellular deformation in odontoblasts. Although odontoblasts detect deformation by the activation of mechanosensitive ionic channels, it is still unclear whether odontoblasts are capable of establishing neurotransmission with myelinated A delta (Aδ) neurons. Additionally, it is still unclear whether these neurons evoke action potentials by neurotransmitters from odontoblasts to mediate sensory transduction in dentin. Thus, we investigated evoked inward currents and evoked action potentials form trigeminal ganglion (TG) neurons after odontoblast mechanical stimulation. We used patch clamp recordings to identify electrophysiological properties and record evoked responses in TG neurons. We classified TG cells into small-sized and medium-sized neurons. In both types of neurons, we observed voltage-dependent inward currents. The currents from medium-sized neurons showed fast inactivation kinetics. When mechanical stimuli were applied to odontoblasts, evoked inward currents were recorded from medium-sized neurons. Antagonists for the ionotropic adenosine triphosphate receptor (P2X 3 ), transient receptor potential channel subfamilies, and Piezo1 channel significantly inhibited these inward currents. Mechanical stimulation to odontoblasts also generated action potentials in the isolectin B 4 -negative medium-sized neurons. Action potentials in these isolectin B 4 -negative medium-sized neurons showed a short duration. Overall, electrophysiological properties of neurons indicate that the TG neurons with recorded evoked responses after odontoblast mechanical stimulation were myelinated Aδ neurons. Odontoblasts established neurotransmission with myelinated Aδ neurons via P2X 3 receptor activation. The results also indicated that mechanosensitive TRP/Piezo1 channels were functionally expressed in odontoblasts. The activation of P2X 3 receptors induced an action potential in the Aδ neurons, underlying a sensory generation mechanism of dentinal pain. Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Calcium channels in solitary retinal ganglion cells from post-natal rat.

PubMed Central

Karschin, A; Lipton, S A

1989-01-01

1. Calcium currents from identified, post-natal retinal ganglion cell neurones from rat were studied with whole-cell and single-channel patch-clamp techniques. Na+ and K+ currents were suppressed with pharmacological agents, allowing isolation of current carried by either 10 mM-Ca2+ or Ba2- during whole-cell recordings. For cell-attached patch recordings, the recording pipette contained 96-110 mM-BaCl2 while the bath solution consisted of isotonic potassium aspartate in order to zero the neuronal membrane potential. 2. A transient component, present in approximately one-third of the whole-cell recordings resembles closely the T-type calcium current observed previously in other tissues. This component activates at low voltages (-40 to -50 mV from holding potentials negative to -80 mV), inactivates with a time constant of 10-30 ms at 35 degrees C, and is carried equally well by Ba2+ or Ca2+. In single-channel recordings small (8 pS) channels are observed whose aggregate microscopic kinetics correspond well to the macroscopic current obtained during whole-cell measurements. 3. During whole-cell recordings, a more prolonged component activates in all retinal ganglion cells at -40 to -20 mV from a holding potential of -90 mV. This component is substantially larger when equimolar Ba2+ replaces Ca2+ as the charge carrier, and is sensitive to the dihydropyridine agonist Bay K8644 (5 microM) and antagonists nifedipine (1-10 microM) and nimodipine (1-10 microM). Thus, the dihydropyridine pharmacology of this prolonged component resembles that of the L-type calcium current found in dorsal root ganglion neurones and in heart cells. Also reminiscent of the L-current, the prolonged component in this preparation is less inactivated at depolarized holding potentials (-60 to -40 mV) than the transient component. In cell-attached recordings, large (20 pS) channels are observed with activation properties similar to those of the prolonged portion of the whole-cell current. 4. omega-Conotoxin fraction GVIA (omega-CgTX VIA), a peptide from the venom of the snail Conus geographus, produces a readily reversible blockade of all components of the calcium current in these central mammalian neurones. This finding is in contrast to that of other preparations in which this toxin is responsible for an ephemeral block of T-current but a long-lasting block of other components of calcium current. 5. In summary, at least two components of calcium current with discrete underlying unitary events are present in post-natal retinal ganglion cells from rat. One component closely resembles the T or transient current observed in other cell types.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2559971

Differences in sodium voltage-gated channel properties according to myosin heavy chain isoform expression in single muscle fibres.

PubMed

Rannou, F; Droguet, M; Giroux-Metges, M A; Pennec, Y; Gioux, M; Pennec, J P

2009-11-01

The myosin heavy chain (MHC) isoform determines the characteristics and shortening velocity of muscle fibres. The functional properties of the muscle fibre are also conditioned by its membrane excitability through the electrophysiological properties of sodium voltage-gated channels. Macropatch-clamp is used to study sodium channels in fibres from peroneus longus (PL) and soleus (Sol) muscles (Wistar rats, n = 8). After patch-clamp recordings, single fibres are identified by SDS-PAGE electrophoresis according to their myosin heavy chain isoform (slow type I and the three fast types IIa, IIx, IIb). Characteristics of sodium currents are compared (Student's t test) between fibres exhibiting only one MHC isoform. Four MHC isoforms are identified in PL and only type I in Sol single fibres. In PL, maximal sodium current (I(max)), maximal sodium conductance (g(Na,max)) and time constants of activation and inactivation ((m) and (h)) increase according to the scheme I-->IIa-->IIx-->IIb (P < 0.05). (m) values related to sodium channel type and/or function, are similar in Sol I and PL IIb fibres (P = 0.97) despite different contractile properties. The voltage dependence of activation (V(a,1/2)) shows a shift towards positive potentials from Sol type I to IIa, IIx and finally IIb fibres from PL (P < 0.05). These data are consistent with the earlier recruitment of slow fibres in a fast-mixed muscle like PL, while slow fibres of postural muscle such as soleus could be recruited in the same ways as IIb fibres in a fast muscle.

Separate Cl^- Conductances Activated by cAMP and Ca2+ in Cl^--Secreting Epithelial Cells

NASA Astrophysics Data System (ADS)

Cliff, William H.; Frizzell, Raymond A.

1990-07-01

We studied the cAMP- and Ca2+-activated secretory Cl^- conductances in the Cl^--secreting colonic epithelial cell line T84 using the whole-cell patch-clamp technique. Cl^- and K^+ currents were measured under voltage clamp. Forskolin or cAMP increased Cl^- current 2-15 times with no change in K^+ current. The current-voltage relation for cAMP-activated Cl^- current was linear from -100 to +100 mV and showed no time-dependent changes in current during voltage pulses. Ca2+ ionophores or increased pipette Ca2+ increased both Cl^- and K^+ currents 2-30 times. The Ca2+-activated Cl^- current was outwardly rectified, activated during depolarizing voltage pulses, and inactivated during hyperpolarizing voltage pulses. Addition of ionophore after forskolin further increased Cl^- conductance 1.5-5 times, and the current took on the time-dependent characteristics of that stimulated by Ca2+. Thus, cAMP and Ca2+ activate Cl^- conductances with different properties, implying that these second messengers activate different Cl^- channels or that they induce different conductive and kinetic states in the same Cl^- channel.

Mechanism of opening a sliding clamp

PubMed Central

Douma, Lauren G.; Yu, Kevin K.; England, Jennifer K.

2017-01-01

Abstract Clamp loaders load ring-shaped sliding clamps onto DNA where the clamps serve as processivity factors for DNA polymerases. In the first stage of clamp loading, clamp loaders bind and stabilize clamps in an open conformation, and in the second stage, clamp loaders place the open clamps around DNA so that the clamps encircle DNA. Here, the mechanism of the initial clamp opening stage is investigated. Mutations were introduced into the Escherichia coli β-sliding clamp that destabilize the dimer interface to determine whether the formation of an open clamp loader–clamp complex is dependent on spontaneous clamp opening events. In other work, we showed that mutation of a positively charged Arg residue at the β-dimer interface and high NaCl concentrations destabilize the clamp, but neither facilitates the formation of an open clamp loader–clamp complex in experiments presented here. Clamp opening reactions could be fit to a minimal three-step ‘bind-open-lock’ model in which the clamp loader binds a closed clamp, the clamp opens, and subsequent conformational rearrangements ‘lock’ the clamp loader–clamp complex in a stable open conformation. Our results support a model in which the E. coli clamp loader actively opens the β-sliding clamp. PMID:28973453

Influence of clamping plate permeability and metal screen structures on three-dimensional magnetic field and eddy current loss in end region of a turbo-generator by numerical analysis

NASA Astrophysics Data System (ADS)

Likun, Wang; Weili, Li; Yi, Xue; Chunwei, Guan

2013-11-01

A significant problem of turbogenerators on complex end structures is overheating of local parts caused by end losses in the end region. Therefore, it is important to investigate the 3-D magnetic field and eddy current loss in the end. In end region of operating large turbogenerator at thermal power plants, magnetic leakage field distribution is complex. In this paper, a 3-D mathematical model used for the calculation of the electromagnetic field in the end region of large turbo-generators is given. The influence of spatial locations of end structures, the actual shape and material of end windings, clamping plate, and copper screen are considered. Adopting the time-step finite element (FE) method and taking the nonlinear characteristics of the core into consideration, a 3-D transient magnetic field is calculated. The objective of this paper is to investigate the influence of clamping plate permeability and metal screen structures on 3-D electromagnetic field distribution and eddy current loss in end region of a turbo-generator. To reduce the temperature of copper screen, a hollow metal screen is proposed. The eddy current loss, which is gained from the 3D transient magnetic field, is used as heat source for the thermal field of end region. The calculated temperatures are compared with test data.

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.

Microelectrode array recordings of cardiac action potentials as a high throughput method to evaluate pesticide toxicity.

PubMed

Natarajan, A; Molnar, P; Sieverdes, K; Jamshidi, A; Hickman, J J

2006-04-01

The threat of environmental pollution, biological warfare agent dissemination and new diseases in recent decades has increased research into cell-based biosensors. The creation of this class of sensors could specifically aid the detection of toxic chemicals and their effects in the environment, such as pyrethroid pesticides. Pyrethroids are synthetic pesticides that have been used increasingly over the last decade to replace other pesticides like DDT. In this study we used a high-throughput method to detect pyrethroids by using multielectrode extracellular recordings from cardiac cells. The data from this cell-electrode hybrid system was compared to published results obtained with patch-clamp electrophysiology and also used as an alternative method to further understand pyrethroid effects. Our biosensor consisted of a confluent monolayer of cardiac myocytes cultured on microelectrode arrays (MEA) composed of 60 substrate-integrated electrodes. Spontaneous activity of these beating cells produced extracellular field potentials in the range of 100 microV to nearly 1200 microV with a beating frequency of 0.5-4 Hz. All of the tested pyrethroids; alpha-Cypermethrin, Tetramethrin and Tefluthrin, produced similar changes in the electrophysiological properties of the cardiac myocytes, namely reduced beating frequency and amplitude. The sensitivity of our toxin detection method was comparable to earlier patch-clamp studies, which indicates that, in specific applications, high-throughput extracellular methods can replace single-cell studies. Moreover, the similar effect of all three pyrethroids on the measured parameters suggests, that not only detection of the toxins but, their classification might also be possible with this method. Overall our results support the idea that whole cell biosensors might be viable alternatives when compared to current toxin detection methods.

Numerical simulation and experimental study of heat-fluid-solid coupling of double flapper-nozzle servo valve

NASA Astrophysics Data System (ADS)

Zhao, Jianhua; Zhou, Songlin; Lu, Xianghui; Gao, Dianrong

2015-09-01

The double flapper-nozzle servo valve is widely used to launch and guide the equipment. Due to the large instantaneous flow rate of servo valve working under specific operating conditions, the temperature of servo valve would reach 120°C and the valve core and valve sleeve deform in a short amount of time. So the control precision of servo valve significantly decreases and the clamping stagnation phenomenon of valve core appears. In order to solve the problem of degraded control accuracy and clamping stagnation of servo valve under large temperature difference circumstance, the numerical simulation of heat-fluid-solid coupling by using finite element method is done. The simulation result shows that zero position leakage of servo valve is basically impacted by oil temperature and change of fit clearance. The clamping stagnation is caused by warpage-deformation and fit clearance reduction of the valve core and valve sleeve. The distribution rules of the temperature and thermal-deformation of shell, valve core and valve sleeve and the pressure, velocity and temperature field of flow channel are also analyzed. Zero position leakage and electromagnet's current when valve core moves in full-stroke are tested using Electro-hydraulic Servo-valve Characteristic Test-bed of an aerospace sciences and technology corporation. The experimental results show that the change law of experimental current at different oil temperatures is roughly identical to simulation current. The current curve of the electromagnet is smooth when oil temperature is below 80°C, but the amplitude of current significantly increases and the hairy appears when oil temperature is above 80°C. The current becomes smooth again after the warped valve core and valve sleeve are reground. It indicates that clamping stagnation is caused by warpage-deformation and fit clearance reduction of valve core and valve sleeve. This paper simulates and tests the heat-fluid-solid coupling of double flapper-nozzle servo valve, and the obtained results provide the reference value for the design of double flapper-nozzle force feedback servo valve.

Action of certain tropine esters on voltage-clamped lobster axon.

PubMed

Blaustein, M P

1968-03-01

Tropine p-tolylacetate (TPTA) and its quaternary analogue, tropine p-tolylacetate methiodide (TPTA MeI) decrease the early transient (Na) and late (K) currents in the voltage-clamped lobster giant axon. These agents, which block the nerve action potential, reduce the maximum Na and K conductance increases associated with membrane depolarization. They also slow the rate at which the sodium conductance is increased and shift the (normalized) membrane conductance vs. voltage curves in the direction of depolarization along the voltage axis. All these effects are qualitatively similar to those resulting from the action of procaine on the voltage-clamped axon. One unusual effect of the tropine esters, noticeable particularly at large depolarization steps, is that they cause the late, K current to reach a peak and then fall off with increasing pulse duration. This effect has not been reported to occur as a result of procaine action. Tropine p-chlorophenyl acetate (TPClphiA), which differs from TPTA only by the substitution of a p-Cl for a p-CH(3) group on the benzene ring, had a negligible effect on axonal excitability.

Action of Certain Tropine Esters on Voltage-Clamped Lobster Axon

PubMed Central

Blaustein, M. P.

1968-01-01

Tropine p-tolylacetate (TPTA) and its quaternary analogue, tropine p-tolylacetate methiodide (TPTA MeI) decrease the early transient (Na) and late (K) currents in the voltage-clamped lobster giant axon. These agents, which block the nerve action potential, reduce the maximum Na and K conductance increases associated with membrane depolarization. They also slow the rate at which the sodium conductance is increased and shift the (normalized) membrane conductance vs. voltage curves in the direction of depolarization along the voltage axis. All these effects are qualitatively similar to those resulting from the action of procaine on the voltage-clamped axon. One unusual effect of the tropine esters, noticeable particularly at large depolarization steps, is that they cause the late, K current to reach a peak and then fall off with increasing pulse duration. This effect has not been reported to occur as a result of procaine action. Tropine p-chlorophenyl acetate (TPClφA), which differs from TPTA only by the substitution of a p-Cl for a p-CH3 group on the benzene ring, had a negligible effect on axonal excitability. PMID:5648830

Adverse impact of intermittent portal clamping on long-term postoperative outcomes in hepatocellular carcinoma.

PubMed

Hao, S; Chen, S; Yang, X; Wan, C

2017-01-01

Introduction To evaluate the impact of intermittent portal clamping (IPC) on long-term postoperative outcomes in patients with hepatocellular carcinoma (HCC). Methods Clinical records of 355 patients underwent curative liver resection for HCC in January 2007 to December 2010 were retrospectively reviewed. According to how portal clamping was performed, patients were grouped as: IPC, n=113; other portal clamping (OPC), n=190; and no portal clamping (NPC), n=52. Results Median recurrence-free survival (RFS) was statistically significantly shorter in the IPC (39.4 months) than OPC (47.3 months, p=0.010) and NPC groups (51.4 months, p=0.008). Median overall survival (OS) was also significantly shorter with IPC (46.3 months), versus 52.9 months with OPC (p=0.022) and 56.2 months with NPC (p=0.015). Kaplan-Meier survival analysis revealed that 5-year cumulative RFS was much lower in the IPC (42.5%) than OPC (50.9%, p=0.014) and NPC groups (49.6%, p=0.013). Five-year cumulative OS was also much lower in the IPC (44.9%) than OPC (58.0%, p=0.020) and NPC groups (57.7%, p=0.025). On univariate analysis, tumour grade, size and number, TNM stage, blood transfusion, vascular invasion and IPC were significantly inversely correlated with RFS and OS. On multivariate analysis, tumour size and number, blood transfusion, vascular invasion and IPC remained significant. Conclusions Our study suggests that IPC is an independent risk factor for poor long-term postoperative outcomes in patients with HCC.

Characterization of bulbospongiosus muscle reflexes activated by urethral distension in male rats.

PubMed

Tanahashi, Masayuki; Karicheti, Venkateswarlu; Thor, Karl B; Marson, Lesley

2012-10-01

The urethrogenital reflex (UGR) is used as a surrogate model of the autonomic and somatic nerve and muscle activity that accompanies ejaculation. The UGR is evoked by distension of the urethra and activation of penile afferents. The current study compares two methods of elevating urethral intraluminal pressure in spinalized, anesthetized male Sprague-Dawley rats (n = 60). The first method, penile extension UGR, involves extracting the penis from the foreskin, so that urethral pressure rises due to a natural anatomical flexure in the penis. The second method, penile clamping UGR, involves penile extension UGR with the addition of clamping of the glans penis. Groups of animals were prepared that either received no additional treatment, surgical shams, or received bilateral nerve cuts (4 nerve cut groups): either the pudendal sensory nerve branch (SbPN), the pelvic nerves, the hypogastric nerves, or all three nerves. Penile clamping UGR was characterized by multiple bursts, monitored by electromyography (EMG) of the bulbospongiosus muscle (BSM) accompanied by elevations in urethral pressure. The penile clamping UGR activity declined across multiple trials and eventually resulted in only a single BSM burst, indicating desensitization. In contrast, the penile extension UGR, without penile clamping, evoked only a single BSM EMG burst that showed no desensitization. Thus, the UGR is composed of two BSM patterns: an initial single burst, termed urethrobulbospongiosus (UBS) reflex and a subsequent multiple bursting pattern (termed ejaculation-like response, ELR) that was only induced with penile clamping urethral occlusion. Transection of the SbPN eliminated the ELR in the penile clamping model, but the single UBS reflex remained in both the clamping and extension models. Pelvic nerve (PelN) transection increased the threshold for inducing BSM activation with both methods of occlusion but actually unmasked an ELR in the penile extension method. Hypogastric nerve (HgN) cuts did not significantly alter any parameter. Transection of all three nerves eliminated BSM activation completely. In conclusion, penile clamping occlusion recruits penile and urethral primary afferent fibers that are necessary for an ELR. Urethral distension without significant penile afferent activation recruits urethral primary afferent fibers carried in either the pelvic or pudendal nerve that are necessary for the single-burst UBS reflex.

[UTP regulates spontaneous transient outward currents in porcine coronary artery smooth muscle cells through PLC-IP(3) signaling pathway].

PubMed

Li, Peng-Yun; Zeng, Xiao-Rong; Yang, Yan; Cai, Fang; Li, Miao-Ling; Liu, Zhi-Fei; Pei, Jie; Zhou, Wen

2008-02-25

The aim of the present study was to investigate the effects of inositol 1,4,5-trisphosphate (IP(3))-generating agonist UTP on spontaneous transient outward currents (STOCs), and explore the role of intracellular Ca(2+) release in the current response mediated by IP(3) in porcine coronary artery smooth muscle cells (CASMCs). The coronary artery was excised from the fresh porcine heart and cut into small segments (2 mm × 5 mm) and then transferred to enzymatic dissociation solution for incubation. Single CASMCs were obtained by two-step enzyme digestion at 37 °C. STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration in freshly isolated porcine CASMCs. The currents were amplified and filtered by patch-clamp amplifier (Axopatch 200B), and then the digitized data were recorded by pClamp 9.0 software and further analyzed by MiniAnalysis 6.0 program. The results were as follows: (1) UTP led to conspicuous increases in STOC amplitude by (57.54±5.34)% and in frequency by (77.46±8.42)% (P<0.01, n=38). (2) The specific blocker of phospholipase C (PLC) - U73122 (5 μmol/L) remarkably reduced STOC amplitude by (31.04±7.46)% and frequency by (41.65±16.59)%, respectively (P<0.05, n=10). In the presence of U73122, UTP failed to reactivate STOCs (n=7). (3) Verapamil (20 μmol/L) and CdCl2 (200 μmol/L), two blockers of L-type voltage-dependent Ca(2+) channels, had little effects on STOCs initiated by UTP (n=8). (4) 1 μmol/L bisindolylmaleimide I (BisI), a potent blocker of protein kinase C (PKC), significantly increased STOC amplitude by (65.44±24.66)% and frequency by (61.35±21.47)% (P<0.01, n=12); UTP (40 μmol/L), applied in the presence of 1 μmol/L BisI, could further increase STOC activity (P<0.05, P<0.01, n=12). Subsequent application of ryanodine (50 μmol/L) abolished STOC activity. (5) In the presence of UTP (40 μmol/L), inhibition of IP(3) receptors (IP(3)Rs) by 2-aminoethoxydiphenyl borate (2-APB, 40 μmol/L) reduced STOC amplitude by (24.08±3.97)% (P<0.05, n=8), but had little effect on STOC frequency (n=8). While application of 2-APB (80 μmol/L) significantly reduced STOC amplitude by (31.43±6.34)% and frequency by (40.59±19.01)%, respectively (P<0.05, P<0.01, n=6). Subsequent application of ryanodine (50 μmol/L) completely blocked STOC activity. Pretreatment of cells with 2-APB (40 μmol/L) or ryanodine (50 μmol/L), UTP (40 μmol/L) failed to reactivate STOCs. The results suggest that UTP activates STOCs mainly via PLC and IP(3)-dependent mechanisms. Complex Ca(2+)-mobilization pathways are involved in UTP-mediated STOC activation in porcine CASMCs.

Testing of Diode-Clamping in an Inductive Pulsed Plasma Thruster Circuit

NASA Technical Reports Server (NTRS)

Toftul, Alexandra; Polzin, Kurt A.; Martin, Adam K.; Hudgins, Jerry L.

2014-01-01

Testing of a 5.5 kV silicon (Si) diode and 5.8 kV prototype silicon carbide (SiC) diode in an inductive pulsed plasma thruster (IPPT) circuit was performed to obtain a comparison of the resulting circuit recapture efficiency,eta(sub r), defined as the percentage of the initial charge energy remaining on the capacitor bank after the diode interrupts the current. The diode was placed in a pulsed circuit in series with a silicon controlled rectifier (SCR) switch, and the voltages across different components and current waveforms were collected over a range of capacitor charge voltages. Reverse recovery parameters, including turn-off time and peak reverse recovery current, were measured and capacitor voltage waveforms were used to determine the recapture efficiency for each case. The Si fast recovery diode in the circuit was shown to yield a recapture efficiency of up to 20% for the conditions tested, while the SiC diode further increased recapture efficiency to nearly 30%. The data presented show that fast recovery diodes operate on a timescale that permits them to clamp the discharge quickly after the first half cycle, supporting the idea that diode-clamping in IPPT circuit reduces energy dissipation that occurs after the first half cycle

Gastrodin Inhibits Allodynia and Hyperalgesia in Painful Diabetic Neuropathy Rats by Decreasing Excitability of Nociceptive Primary Sensory Neurons

PubMed Central

Ye, Xin; Han, Wen-Juan; Wang, Wen-Ting; Luo, Ceng; Hu, San-Jue

2012-01-01

Painful diabetic neuropathy (PDN) is a common complication of diabetes mellitus and adversely affects the patients’ quality of life. Evidence has accumulated that PDN is associated with hyperexcitability of peripheral nociceptive primary sensory neurons. However, the precise cellular mechanism underlying PDN remains elusive. This may result in the lacking of effective therapies for the treatment of PDN. The phenolic glucoside, gastrodin, which is a main constituent of the Chinese herbal medicine Gastrodia elata Blume, has been widely used as an anticonvulsant, sedative, and analgesic since ancient times. However, the cellular mechanisms underlying its analgesic actions are not well understood. By utilizing a combination of behavioral surveys and electrophysiological recordings, the present study investigated the role of gastrodin in an experimental rat model of STZ-induced PDN and to further explore the underlying cellular mechanisms. Intraperitoneal administration of gastrodin effectively attenuated both the mechanical allodynia and thermal hyperalgesia induced by STZ injection. Whole-cell patch clamp recordings were obtained from nociceptive, capsaicin-sensitive small diameter neurons of the intact dorsal root ganglion (DRG). Recordings from diabetic rats revealed that the abnormal hyperexcitability of neurons was greatly abolished by application of GAS. To determine which currents were involved in the antinociceptive action of gastrodin, we examined the effects of gastrodin on transient sodium currents (I NaT) and potassium currents in diabetic small DRG neurons. Diabetes caused a prominent enhancement of I NaT and a decrease of potassium currents, especially slowly inactivating potassium currents (I AS); these effects were completely reversed by GAS in a dose-dependent manner. Furthermore, changes in activation and inactivation kinetics of I NaT and total potassium current as well as I AS currents induced by STZ were normalized by GAS. This study provides a clear cellular basis for the peripheral analgesic action of gastrodin for the treatment of chronic pain, including PDN. PMID:22761855

Modulation of hypoglossal motoneuron excitability by NK1 receptor activation in neonatal mice in vitro

PubMed Central

Yasuda, Kouichi; Robinson, Dean M; Selvaratnam, Subramaniam R; Walsh, Carmen W; McMorland, Angus J C; Funk, Gregory D

2001-01-01

The effects of substance P (SP), acting at NK1 receptors, on the excitability and inspiratory activity of hypoglossal (XII) motoneurons (MNs) were investigated using rhythmically active medullary-slice preparations from neonatal mice (postnatal day 0–3). Local application of the NK1 agonist [SAR9,Met (O2)11]-SP (SPNK1) produced a dose-dependent, spantide- (a non-specific NK receptor antagonist) and GR82334-(an NK1 antagonist) sensitive increase in inspiratory burst amplitude recorded from XII nerves. Under current clamp, SPNK1 significantly depolarized XII MNs, potentiated repetitive firing responses to injected currents and produced a leftward shift in the firing frequency-current relationships without affecting slope. Under voltage clamp, SPNK1 evoked an inward current and increased input resistance, but had no effect on inspiratory synaptic currents. SPNK1 currents persisted in the presence of TTX, were GR82334 sensitive, were reduced with hyperpolarization and reversed near the expected EK. Effects of the α1-noradrenergic receptor agonist phenylephrine (PE) on repetitive firing behaviour were virtually identical to those of SPNK1. Moreover, SPNK1 currents were completely occluded by PE, suggesting that common intracellular pathways mediate the actions of NK1 and α1-noradrenergic receptors. In spite of the similar actions of SPNK1 and PE on XII MN responses to somally injected current, α1-noradrenergic receptor activation potentiated inspiratory synaptic currents and was more than twice as effective in potentiating XII nerve inspiratory burst amplitude. GR82334 reduced XII nerve inspiratory burst amplitude and generated a small outward current in XII MNs. These observations, together with the first immunohistochemical evidence in the newborn for SP immunopositive terminals in the vicinity of SPNK1-sensitive inspiratory XII MNs, support the endogenous modulation of XII MN excitability by SP. In contrast to phrenic MNs (Ptak et al. 2000), blocking NMDA receptors with AP5 had no effect on the modulation of XII nerve activity by SPNK1. In conclusion, SPNK1 modulates XII motoneuron responses to inspiratory drive primarily through inhibition of a resting, postsynaptic K+ leak conductance. The results establish the functional significance of SP in controlling upper airway tone during early postnatal life and indicate differential modulation of motoneurons controlling airway and pump muscles by SP. PMID:11454963

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.

Fibrin Sealant Improves Hemostasis in Peripheral Vascular Surgery: A Randomized Prospective Trial

PubMed Central

Schenk, Worthington G.; Burks, Sandra G.; Gagne, Paul J.; Kagan, Steven A.; Lawson, Jeffrey H.; Spotnitz, William D.

2003-01-01

Objective To evaluate the efficacy and safety of an investigational fibrin sealant (FS) in a randomized prospective, partially blinded, controlled, multicenter trial. Summary Background Data Upper extremity vascular access surgery using polytetrafluorethylene (PTFE) graft placement for dialysis was chosen as a reproducible, clinically relevant model for evaluating the usefulness of FS. The FS consisted of pooled human fibrinogen (60 mg/mL) and thrombin (500 NIH U/mL). Time to hemostasis was measured, and adverse events were monitored. Methods Consenting adult patients (n = 48) undergoing placement of a standard PTFE graft were randomized in a 2:1:1 ratio to the treatment group using FS (ZLB Bioplasma AG, Bern, Switzerland), oxidized regenerated cellulose (Surgicel, Johnson & Johnson, New Brunswick, NJ), or pressure. Patients received heparin (3,000 IU IVP) before placement of vascular clamps. If the treatment was FS, clamps were left in place for 120 seconds after the application of study material to permit polymerization. If treatment was Surgicel, clamps were left in place until the agent had been applied according to manufacturer’s instructions. If the treatment was pressure, clamps were released as soon as the investigator was ready to apply compression. Immediately after release of the last clamp, the arterial and venous suture lines were evaluated for bleeding. The time to hemostasis at both the venous and arterial sites was recorded. Results Significant (P ≤ .005) reduction in time to hemostasis was achieved in the FS group. Thirteen (54.2%) patients randomized to FS experienced immediate hemostasis at both suture lines following clamp removal compared to no patients using Surgicel or pressure. Only one patient (7.1%) in the Surgicel group and no patients in the pressure group experienced hemostasis at 120 seconds from clamp removal, compared to 13 (54.2%) patients for FS. Adverse events were comparable in all groups. There were no seroconversions. Conclusions FS achieved more rapid hemostasis than traditional techniques in this peripheral vascular procedure. FS use appeared to be safe for this procedure. PMID:12796584

[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.

Faster than the Speed of Hearing: Nanomechanical Force Probes Enable the Electromechanical Observation of Cochlear Hair Cells

PubMed Central

Doll, Joseph C.; Peng, Anthony W.; Ricci, Anthony J.; Pruitt, Beth L.

2012-01-01

Understanding the mechanisms responsible for our sense of hearing requires new tools for unprecedented stimulation and monitoring of sensory cell mechanotransduction at frequencies yet to be explored. We describe nanomechanical force probes designed to evoke mechanotransduction currents at up to 100kHz in living cells. High-speed force and displacement metrology is enabled by integrating piezoresistive sensors and piezoelectric actuators onto nanoscale cantilevers. The design, fabrication process, actuator performance and actuator-sensor crosstalk compensation results are presented. We demonstrate the measurement of mammalian cochlear hair cell mechanotransduction with simultaneous patch clamp recordings at unprecedented speeds. The probes can deliver mechanical stimuli with sub-10 μs rise times in water and are compatible with standard upright and inverted microscopes. PMID:23181721

Epidermal growth factor upregulates motility of Mat-LyLu rat prostate cancer cells partially via voltage-gated Na+ channel activity

PubMed Central

Ding, Yanning; Brackenbury, William J.; Onganer, Pinar U.; Montano, Ximena; Porter, Louise M.; Bates, Lucy F.; Djamgoz, Mustafa B. A.

2014-01-01

The main aim of this investigation was to determine whether a functional relationship existed between epidermal growth factor (EGF) and voltage-gated sodium channel (VGSC) upregulation, both associated with strongly metastatic prostate cancer cells. Incubation with EGF for 24 h more than doubled VGSC current density. Similar treatment with EGF significantly and dose-dependently enhanced the cells’ migration through Transwell filters. Both the patch clamp recordings and the migration assay suggested that endogenous EGF played a similar role. Importantly, co-application of EGF and tetrodotoxin, a highly selective VGSC blocker, abolished 65% of the potentiating effect of EGF. It is suggested that a significant portion of the EGF-induced enhancement of migration occurred via VGSC activity. PMID:17960590

Photoelectrochemical modulation of neuronal activity with free-standing coaxial silicon nanowires

NASA Astrophysics Data System (ADS)

Parameswaran, Ramya; Carvalho-de-Souza, João L.; Jiang, Yuanwen; Burke, Michael J.; Zimmerman, John F.; Koehler, Kelliann; Phillips, Andrew W.; Yi, Jaeseok; Adams, Erin J.; Bezanilla, Francisco; Tian, Bozhi

2018-02-01

Optical methods for modulating cellular behaviour are promising for both fundamental and clinical applications. However, most available methods are either mechanically invasive, require genetic manipulation of target cells or cannot provide subcellular specificity. Here, we address all these issues by showing optical neuromodulation with free-standing coaxial p-type/intrinsic/n-type silicon nanowires. We reveal the presence of atomic gold on the nanowire surfaces, likely due to gold diffusion during the material growth. To evaluate how surface gold impacts the photoelectrochemical properties of single nanowires, we used modified quartz pipettes from a patch clamp and recorded sustained cathodic photocurrents from single nanowires. We show that these currents can elicit action potentials in primary rat dorsal root ganglion neurons through a primarily atomic gold-enhanced photoelectrochemical process.

Slowly inactivating component of Na+ current in peri-somatic region of hippocampal CA1 pyramidal neurons

PubMed Central

Park, Yul Young; Johnston, Daniel

2013-01-01

The properties of voltage-gated ion channels on the neuronal membrane shape electrical activity such as generation and backpropagation of action potentials, initiation of dendritic spikes, and integration of synaptic inputs. Subthreshold currents mediated by sodium channels are of interest because of their activation near rest, slow inactivation kinetics, and consequent effects on excitability. Modulation of these currents can also perturb physiological responses of a neuron that might underlie pathological states such as epilepsy. Using nucleated patches from the peri-somatic region of hippocampal CA1 neurons, we recorded a slowly inactivating component of the macroscopic Na+ current (which we have called INaS) that shared many biophysical properties with the persistent Na+ current, INaP, but showed distinctively faster inactivating kinetics. Ramp voltage commands with a velocity of 400 mV/s were found to elicit this component of Na+ current reliably. INaS also showed a more hyperpolarized I-V relationship and slower inactivation than those of the fast transient Na+ current (INaT) recorded in the same patches. The peak amplitude of INaS was proportional to the peak amplitude of INaT but was much smaller in amplitude. Hexanol, riluzole, and ranolazine, known Na+ channel blockers, were tested to compare their effects on both INaS and INaT. The peak conductance of INaS was preferentially blocked by hexanol and riluzole, but the shift of half-inactivation voltage (V1/2) was only observed in the presence of riluzole. Current-clamp measurements with hexanol suggested that INaS was involved in generation of an action potential and in upregulation of neuronal excitability. PMID:23236005

[Decreased A-type potassium current mediates the hyperexcitability of nociceptive neurons in the chronically compressed dorsal root ganglia].

PubMed

Yan, Ni; Li, Xiao-Han; Cheng, Qi; Yan, Jin; Ni, Xin; Sun, Ji-Hu

2007-04-25

The excitability of nociceptive neurons increases in the intact dorsal root ganglion (DRG) after a chronic compression, but the underlying mechanisms are still unclear. The aim of this study was to investigate the ionic mechanisms underlying the hyperexcitability of nociceptive neurons in the compressed ganglion. Chronic compression of DRG (CCD) was produced in adult rats by inserting two rods through the intervertebral foramina to compress the L4 DRG and the ipsilateral L5 DRG. After 5-7 d, DRG somata were dissociated and placed in culture for 12-18 h. In sharp electrode recording model, the lower current threshold and the depolarized membrane potential in the acutely dissociated CCD neurons were detected, indicating that hyperexcitability is intrinsic to the soma. Since voltage-gated K(+) (Kv) channels in the primary sensory neurons are important for the regulation of excitability, we hypothesized that CCD would alter K(+) current properties in the primary sensory neurons. We examined the effects of 4-aminopyridine (4-AP), a specific antagonist of A-type potassium channel, on the excitability of the control DRG neurons. With 4-AP in the external solution, the control DRG neurons depolarized (with discharges in some cells) and their current threshold decreased as the CCD neurons demonstrated, indicating the involvement of decreased A-type potassium current in the hyperexcitability of the injured neurons. Furthermore, the alteration of A-type potassium current in nociceptive neurons in the compressed ganglion was investigated with the whole-cell patch-clamp recording model. CCD significantly decreased A-type potassium current density in nociceptive DRG neurons. These data suggest that a reduction in A-type potassium current contributes, at least in part, to the increase in neuron excitability that may lead to the development of pain and hyperalgesia associated with CCD.

Inhibition of recombinant Ca(v)3.1 (alpha(1G)) T-type calcium channels by the antipsychotic drug clozapine.

PubMed

Choi, Kee-Hyun; Rhim, Hyewhon

2010-01-25

Low voltage-activated T-type calcium channels are involved in the regulation of the neuronal excitability, and could be subject to many antipsychotic drugs. The effects of clozapine, an atypical antipsychotic drug, on recombinant Ca(v)3.1 T-type calcium channels heterologously expressed in human embryonic kidney 293 cells were examined using whole-cell patch-clamp recordings. At a standard holding potential of -100 mV, clozapine inhibited Ca(v)3.1 currents with an IC(50) value of 23.7+/-1.3 microM in a use-dependent manner. However, 10 microM clozapine inhibited more than 50% of the Ca(v)3.1 currents in recordings at a more physiologically relevant holding potential of -75 mV. Clozapine caused a significant hyperpolarizing shift in the steady-state inactivation curve of the Ca(v)3.1 channels, which is presumably the main mechanism accounting for the inhibition of the Ca(v)3.1 currents. In addition, clozapine slowed Ca(v)3.1 deactivation and inactivation kinetics but not activation kinetics. Clozapine-induced changes in deactivation and inactivation rates of the Ca(v)3.1 channel gating would likely facilitate calcium influx via Ca(v)3.1 T-type calcium channels. Thus, clozapine may exert its therapeutic and/or side effects by altering cell's excitability and firing properties through actions on T-type calcium channels.

In-situ recording of ionic currents in projection neurons and Kenyon cells in the olfactory pathway of the honeybee

PubMed Central

Rössler, Wolfgang

2018-01-01

The honeybee olfactory pathway comprises an intriguing pattern of convergence and divergence: ~60.000 olfactory sensory neurons (OSN) convey olfactory information on ~900 projection neurons (PN) in the antennal lobe (AL). To transmit this information reliably, PNs employ relatively high spiking frequencies with complex patterns. PNs project via a dual olfactory pathway to the mushroom bodies (MB). This pathway comprises the medial (m-ALT) and the lateral antennal lobe tract (l-ALT). PNs from both tracts transmit information from a wide range of similar odors, but with distinct differences in coding properties. In the MBs, PNs form synapses with many Kenyon cells (KC) that encode odors in a spatially and temporally sparse way. The transformation from complex information coding to sparse coding is a well-known phenomenon in insect olfactory coding. Intrinsic neuronal properties as well as GABAergic inhibition are thought to contribute to this change in odor representation. In the present study, we identified intrinsic neuronal properties promoting coding differences between PNs and KCs using in-situ patch-clamp recordings in the intact brain. We found very prominent K+ currents in KCs clearly differing from the PN currents. This suggests that odor coding differences between PNs and KCs may be caused by differences in their specific ion channel properties. Comparison of ionic currents of m- and l-ALT PNs did not reveal any differences at a qualitative level. PMID:29351552

Automatic parameter estimation of multicompartmental neuron models via minimization of trace error with control adjustment.

PubMed

Brookings, Ted; Goeritz, Marie L; Marder, Eve

2014-11-01

We describe a new technique to fit conductance-based neuron models to intracellular voltage traces from isolated biological neurons. The biological neurons are recorded in current-clamp with pink (1/f) noise injected to perturb the activity of the neuron. The new algorithm finds a set of parameters that allows a multicompartmental model neuron to match the recorded voltage trace. Attempting to match a recorded voltage trace directly has a well-known problem: mismatch in the timing of action potentials between biological and model neuron is inevitable and results in poor phenomenological match between the model and data. Our approach avoids this by applying a weak control adjustment to the model to promote alignment during the fitting procedure. This approach is closely related to the control theoretic concept of a Luenberger observer. We tested this approach on synthetic data and on data recorded from an anterior gastric receptor neuron from the stomatogastric ganglion of the crab Cancer borealis. To test the flexibility of this approach, the synthetic data were constructed with conductance models that were different from the ones used in the fitting model. For both synthetic and biological data, the resultant models had good spike-timing accuracy. Copyright © 2014 the American Physiological Society.

An equivalent circuit for small atrial trabeculae of frog.

PubMed Central

Jakobsson, E; Barr, L; Connor, J A

1975-01-01

An equivalent electrical circuit has been constructed for small atrial trabecula of frog in a double sucrose gap voltage clamp apparatus. The basic strategy in constructing the circuit was to derive the distribution of membrane capacitance and extracellular resistance from the preparation's response to small voltage displacements near the resting condition, when the membrane conductance is presumably quite low. Then standard Hodgkin-Huxley channels were placed in parallel with the capacitance and the results of voltage clamp experiments were simulated. The results suggest that the membranes of the preparation cannot in fact be clamped near the control voltage nor can the ionic currents be measured directly with reasonable accuracy by axon standards. It may or may not be a realizable goal in the future to define the preparation's electrical behavior well enough to permit the ultimate quantitative description of the membrane's specific ion conductances. The result of this paper suggest that if this goal is achieved using the double sucrose gap voltage clamp, it will be by a detailed quantitative accounting for substantial irreducible errors in voltage control, rather than by experimental achievement of good voltage control. PMID:1203441

Ion pathways in the taste bud and their significance for transduction.

PubMed

DeSimone, J A; Ye, Q; Heck, G L

1993-01-01

Taste buds share a topology with ion-transporting epithelial and evidence now indicates that neural responses in rats to Na+ salts of differing anion are mediated by both transcellular and paracellular ion transport. Na+ exerts its effects mainly on the transcellular pathway. Neural responses to Na+ salts are enhanced by negative voltage clamp and suppressed by positive clamp in a manner indicating modulation of the apical membrane potential of receptor cells. Anion effects are mainly paracellular. Under zero current clamp increasing anion size reduces the neural response at constant Na+ concentration. Below about 50 mM this difference is entirely eliminated under voltage clamp. This suggests that paracellular transepithelial potentials normally create an anion difference. At higher concentrations the relatively high permeability of the paracellular shunt to Cl- permits sufficient electroneutral diffusion of NaCl below the tight junctions to stimulate cells that do not make direct contact with the oral cavity. In general, the sensitivity of a response to perturbations in the apical membrane potential indicates that some phase of Na+ salt taste transduction is accompanied by changes in an apical membrane channel conductance.

IS IT TIME TO RETHINK CORD MANAGEMENT WHEN RESUSCITATION IS NEEDED?

PubMed Central

Mercer, Judith S.; Erickson-Owens, Debra A.

2015-01-01

An infant who receives a placental transfusion at birth, either from cord milking or delayed cord clamping, obtains about 30% more blood volume than the infant whose cord is cut immediately. Receiving an adequate blood volume from placental transfusion at birth may be protective for the distressed neonate as it prevents hypovolemia and can support optimal perfusion to all organs. New research shows that ventilating before clamping the umbilical cord can reduce large swings in cardiovascular function and help to stabilize the infant. Hypovolemia, often associated with nuchal cord or shoulder dystocia, may lead to an inflammatory cascade and subsequent ischemic injury. A sudden unexpected neonatal asystole at birth may occur from severe hypovolemia. The restoration of blood volume is an important action to protect the hearts and brains of these neonates. Current protocols for resuscitation imply immediate cord clamping and the care of the infant away from the mother's bedside. We suggest that an obstetrical provider can achieve placental transfusion for the distressed neonate by milking the cord several times or resuscitating the infant at the perineum with an intact cord. Milking the cord can be done quickly within the current Neonatal Resuscitation Program guidelines. Cord blood gases can be collected with delayed cord clamping. “Bringing the resuscitation” to the mother's bedside is a novel concept and supports an intact cord. Adopting a policy for resuscitation with an intact cord in a hospital setting will take concentrated effort and team work by obstetrics, pediatrics, midwifery, and nursing. PMID:25297530

Intrinsic and synaptic properties of vertical cells of the mouse dorsal cochlear nucleus

PubMed Central

Kuo, Sidney P.; Lu, Hsin-Wei

2012-01-01

Multiple classes of inhibitory interneurons shape the activity of principal neurons of the dorsal cochlear nucleus (DCN), a primary target of auditory nerve fibers in the mammalian brain stem. Feedforward inhibition mediated by glycinergic vertical cells (also termed tuberculoventral or corn cells) is thought to contribute importantly to the sound-evoked response properties of principal neurons, but the cellular and synaptic properties that determine how vertical cells function are unclear. We used transgenic mice in which glycinergic neurons express green fluorescent protein (GFP) to target vertical cells for whole cell patch-clamp recordings in acute slices of DCN. We found that vertical cells express diverse intrinsic spiking properties and could fire action potentials at high, sustained spiking rates. Using paired recordings, we directly examined synapses made by vertical cells onto fusiform cells, a primary DCN principal cell type. Vertical cell synapses produced unexpectedly small-amplitude unitary currents in fusiform cells, and additional experiments indicated that multiple vertical cells must be simultaneously active to inhibit fusiform cell spike output. Paired recordings also revealed that a major source of inhibition to vertical cells comes from other vertical cells. PMID:22572947

Functional Na+ Channels in Cell Adhesion probed by Transistor Recording

PubMed Central

Schmidtner, Markus; Fromherz, Peter

2006-01-01

Cell membranes in a tissue are in close contact to each other, embedded in the extracellular matrix. Standard electrophysiological methods are not able to characterize ion channels under these conditions. Here we consider the area of cell adhesion on a solid substrate as a model system. We used HEK 293 cells cultured on fibronectin and studied the activation of NaV1.4 sodium channels in the adherent membrane with field-effect transistors in a silicon substrate. Under voltage clamp, we compared the transistor response with the whole-cell current. We observed that the extracellular voltage in the cell-chip contact was proportional to the total membrane current. The relation was calibrated by alternating-current stimulation. We found that Na+ channels are present in the area of cell adhesion on fibronectin with a functionality and a density that is indistinguishable from the free membrane. The experiment provides a basis for studying selective accumulation and depletion of ion channels in cell adhesion and also for a development of cell-based biosensoric devices and neuroelectronic systems. PMID:16227504

Modulation of voltage-gated conductances of retinal horizontal cells by UV-excited TiO2 nanoparticles.

PubMed

Meshik, Xenia; Choi, Min; Baker, Adam; Malchow, R Paul; Covnot, Leigha; Doan, Samuel; Mukherjee, Souvik; Farid, Sidra; Dutta, Mitra; Stroscio, Michael A

2017-04-01

This study examines the ability of optically-excited titanium dioxide nanoparticles to influence voltage-gated ion channels in retinal horizontal cells. Voltage clamp recordings were obtained in the presence and absence of TiO 2 and ultraviolet laser excitation. Significant current changes were observed in response to UV light, particularly in the -40 mV to +40 mV region where voltage-gated Na + and K + channels have the highest conductance. Cells in proximity to UV-excited TiO 2 exhibited a left-shift in the current-voltage relation of around 10 mV in the activation of Na + currents. These trends were not observed in control experiments where cells were excited with UV light without being exposed to TiO 2 . Electrostatic force microscopy confirmed that electric fields can be induced in TiO 2 with UV light. Simulations using the Hodgkin-Huxley model yielded results which agreed with the experimental data and showed the I-V characteristics of individual ion channels in the presence of UV-excited TiO 2 . Copyright © 2016 Elsevier Inc. All rights reserved.

De novo mutations in HCN1 cause early infantile epileptic encephalopathy.

PubMed

Nava, Caroline; Dalle, Carine; Rastetter, Agnès; Striano, Pasquale; de Kovel, Carolien G F; Nabbout, Rima; Cancès, Claude; Ville, Dorothée; Brilstra, Eva H; Gobbi, Giuseppe; Raffo, Emmanuel; Bouteiller, Delphine; Marie, Yannick; Trouillard, Oriane; Robbiano, Angela; Keren, Boris; Agher, Dahbia; Roze, Emmanuel; Lesage, Suzanne; Nicolas, Aude; Brice, Alexis; Baulac, Michel; Vogt, Cornelia; El Hajj, Nady; Schneider, Eberhard; Suls, Arvid; Weckhuysen, Sarah; Gormley, Padhraig; Lehesjoki, Anna-Elina; De Jonghe, Peter; Helbig, Ingo; Baulac, Stéphanie; Zara, Federico; Koeleman, Bobby P C; Haaf, Thomas; LeGuern, Eric; Depienne, Christel

2014-06-01

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contribute to cationic Ih current in neurons and regulate the excitability of neuronal networks. Studies in rat models have shown that the Hcn1 gene has a key role in epilepsy, but clinical evidence implicating HCN1 mutations in human epilepsy is lacking. We carried out exome sequencing for parent-offspring trios with fever-sensitive, intractable epileptic encephalopathy, leading to the discovery of two de novo missense HCN1 mutations. Screening of follow-up cohorts comprising 157 cases in total identified 4 additional amino acid substitutions. Patch-clamp recordings of Ih currents in cells expressing wild-type or mutant human HCN1 channels showed that the mutations had striking but divergent effects on homomeric channels. Individuals with mutations had clinical features resembling those of Dravet syndrome with progression toward atypical absences, intellectual disability and autistic traits. These findings provide clear evidence that de novo HCN1 point mutations cause a recognizable early-onset epileptic encephalopathy in humans.

Deployment of the Oklahoma borehole seismic experiment

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

Harben, P.E.; Rock, D.W.

1989-01-20

This paper discusses the Oklahoma borehole seismic experiment, currently in operation, set up by members of the Lawrence Livermore National Laboratory Treaty Verification Program and the Oklahoma Geophysical Observatory to determine deep-borehole seismic characteristics in geology typical of large regions in the Soviet Union. We evaluated and logged an existing 772-m deep borehole on the Observatory site by running caliper, cement bonding, casing inspection, and hole-deviation logs. Two Teledyne Geotech borehole-clamping seismometers were placed at various depths and spacings in the deep borehole. Currently, they are deployed at 727 and 730 m. A Teledyne Geotech shallow-borehole seismometer was mounted inmore » a 4.5-m hole, one meter from the deep borehole. The seismometers' system coherency were tested and found to be excellent to 35 Hz. We have recorded seismic noise, quarry blasts, regional earthquakes and teleseisms in the present configuration. We will begin a study of seismic noise and attenuation as a function of depth in the near future. 7 refs., 18 figs.« less

Contextual fear conditioning depresses infralimbic excitability.

PubMed

Soler-Cedeño, Omar; Cruz, Emmanuel; Criado-Marrero, Marangelie; Porter, James T

2016-04-01

Patients with posttraumatic stress disorder (PTSD) show hypo-active ventromedial prefrontal cortices (vmPFC) that correlate with their impaired ability to discriminate between safe and dangerous contexts and cues. Previously, we found that auditory fear conditioning depresses the excitability of neurons populating the homologous structure in rodents, the infralimbic cortex (IL). However, it is undetermined if IL depression was mediated by the cued or contextual information. The objective of this study was to examine whether contextual information was sufficient to depress IL neuronal excitability. After exposing rats to context-alone, pseudoconditioning, or contextual fear conditioning, we used whole-cell current-clamp recordings to examine the excitability of IL neurons in prefrontal brain slices. We found that contextual fear conditioning reduced IL neuronal firing in response to depolarizing current steps. In addition, neurons from contextual fear conditioned animals showed increased slow afterhyperpolarization potentials (sAHPs). Moreover, the observed changes in IL excitability correlated with contextual fear expression, suggesting that IL depression may contribute to the encoding of contextual fear. Copyright © 2016 Elsevier Inc. All rights reserved.

Rate dependency of delayed rectifier currents during the guinea-pig ventricular action potential

PubMed Central

Rocchetti, Marcella; Besana, Alessandra; Gurrola, Georgina B; Possani, Lourival D; Zaza, Antonio

2001-01-01

The action potential clamp technique was exploited to evaluate the rate dependency of delayed rectifier currents (IKr and IKs) during physiological electrical activity. IKr and IKs were measured in guinea-pig ventricular myocytes at pacing cycle lengths (CL) of 1000 and 250 ms.A shorter CL, with the attendant changes in action potential shape, was associated with earlier activation and increased magnitude of both IKr and IKs. Nonetheless, the relative contributions of IKr and IKs to total transmembrane current were independent of CL.Shortening of diastolic interval only (constant action potential shape) enhanced IKs, but not IKr.IKr was increased by a change in the action potential shape only (constant diastolic interval).In ramp clamp experiments, IKr amplitude was directly proportional to repolarization rate at values within the low physiological range (< 1.0 V s−1); at higher repolarization rates proportionality became shallower and finally reversed.When action potential duration (APD) was modulated by constant current injection (I-clamp), repolarization rates > 1.0 V s−1 were associated with a reduced effect of IKr block on APD. The effect of changes in repolarization rate was independent of CL and occurred in the presence of IKs blockade.In spite of its complexity, the behaviour of IKr was accurately predicted by a numerical model based entirely on known kinetic properties of the current.Both IKr and IKs may be increased at fast heart rates, but this may occur through completely different mechanisms. The mechanisms identified are such as to contribute to abnormal rate dependency of repolarization in prolonged repolarization syndromes. PMID:11483703

Is early cord clamping, delayed cord clamping or cord milking best?

PubMed

Vatansever, Binay; Demirel, Gamze; Ciler Eren, Elif; Erel, Ozcan; Neselioglu, Salim; Karavar, Hande Nur; Gundogdu, Semra; Ulfer, Gozde; Bahadir, Selcen; Tastekin, Ayhan

2018-04-01

To compare the antioxidant status of three cord clamping procedures (early clamping, delayed clamping and milking) by analyzing the thiol-disulfide balance. This randomized controlled study enrolled 189 term infants who were divided into three groups according to the cord clamping procedure: early clamping, delayed clamping and milking. Blood samples were collected from the umbilical arteries immediately after clamping, and the thiol/disulfide homeostasis was analyzed. The native and total thiol levels were significantly (p < .05) lower in the early cord clamping group compared with the other two groups. The disulfide/total thiol ratio was significantly (p = .026) lower in the delayed cord clamping and milking groups compared with the early clamping groups. Early cord clamping causes the production of more disulfide bonds and lower thiol levels, indicating that oxidation reactions are increased in the early cord clamping procedure compared with the delayed cord clamping and milking procedures. The oxidant capacity is greater with early cord clamping than with delayed clamping or cord milking. Delayed cord clamping or milking are beneficial in neonatal care, and we suggest that they be performed routinely in all deliveries.

Active signal conduction through the sensory dendrite of a spider mechanoreceptor neuron.

PubMed

Gingl, Ewald; French, Andrew S

2003-07-09

Rapid responses to sensory stimulation are crucial for survival. This must be especially true for mechanical stimuli containing temporal information, such as vibration. Sensory transduction occurs at the tips of relatively long sensory dendrites in many mechanoreceptors of both vertebrates and invertebrates, but little is known about the electrical properties of these crucial links between transduction and action potential generation. The VS-3 slit-sense organ of the spider Cupiennius salei contains bipolar mechanosensory neurons that allow voltage-clamp recording from the somata, whereas mechanotransduction occurs at the tips of 100- to 200-microm-long sensory dendrites. We studied the properties of VS-3 sensory dendrites using three approaches. Voltage-jump experiments measured the spread of voltage outward from the soma by observing total mechanically transduced charge recovered at the soma as a function of time after a voltage jump. Frequency-response measurements between pseudorandom mechanical stimulation and somatic membrane potential estimated the passive cable properties of the dendrite for voltage spread in the opposite direction. Both of these sets of data indicated that the dendritic cable would significantly attenuate and retard a passively propagated receptor potential. Finally, current-clamp observations of receptor potentials and action potentials indicated that action potentials normally start at the distal dendrites and propagate regeneratively to the soma, reducing the temporal delay of passive conduction.

High-voltage-activated calcium current subtypes in mouse DRG neurons adapt in a subpopulation-specific manner after nerve injury.

PubMed

Murali, Swetha S; Napier, Ian A; Mohammadi, Sarasa A; Alewood, Paul F; Lewis, Richard J; Christie, MacDonald J

2015-03-01

Changes in ion channel function and expression are characteristic of neuropathic pain. Voltage-gated calcium channels (VGCCs) are integral for neurotransmission and membrane excitability, but relatively little is known about changes in their expression after nerve injury. In this study, we investigate whether peripheral nerve ligation is followed by changes in the density and proportion of high-voltage-activated (HVA) VGCC current subtypes in dorsal root ganglion (DRG) neurons, the contribution of presynaptic N-type calcium channels in evoked excitatory postsynaptic currents (EPSCs) recorded from dorsal horn neurons in the spinal cord, and the changes in expression of mRNA encoding VGCC subunits in DRG neurons. Using C57BL/6 mice [8- to 11-wk-old males (n = 91)] for partial sciatic nerve ligation or sham surgery, we performed whole cell patch-clamp recordings on isolated DRG neurons and dorsal horn neurons and measured the expression of all VGCC subunits with RT-PCR in DRG neurons. After nerve injury, the density of P/Q-type current was reduced overall in DRG neurons. There was an increase in the percentage of N-type and a decrease in that of P/Q-type current in medium- to large-diameter neurons. No changes were found in the contribution of presynaptic N-type calcium channels in evoked EPSCs recorded from dorsal horn neurons. The α2δ-1 subunit was upregulated by 1.7-fold and γ-3, γ-2, and β-4 subunits were all downregulated 1.7-fold in injured neurons compared with sham-operated neurons. This comprehensive characterization of HVA VGCC subtypes in mouse DRG neurons after nerve injury revealed changes in N- and P/Q-type current proportions only in medium- to large-diameter neurons. Copyright © 2015 the American Physiological Society.

Effects of allocryptopine on outward potassium current and slow delayed rectifier potassium current in rabbit myocardium.

PubMed

Fu, Yi-Cheng; Zhang, Yu; Tian, Liu-Yang; Li, Nan; Chen, Xi; Cai, Zhong-Qi; Zhu, Chao; Li, Yang

2016-05-01

Allocryptopine (ALL) is an effective alkaloid of Corydalis decumbens (Thunb.) Pers. Papaveraceae and has proved to be anti-arrhythmic. The purpose of our study is to investigate the effects of ALL on transmural repolarizing ionic ingredients of outward potassium current (I to) and slow delayed rectifier potassium current (I Ks). The monophasic action potential (MAP) technique was used to record the MAP duration of the epicardium (Epi), myocardium (M) and endocardium (Endo) of the rabbit heart and the whole cell patch clamp was used to record I to and I Ks in cardiomyocytes of Epi, M and Endo layers that were isolated from rabbit ventricles. The effects of ALL on MAP of Epi, M and Endo layers were disequilibrium. ALL could effectively reduce the transmural dispersion of repolarization (TDR) in rabbit transmural ventricular wall. ALL decreased the current densities of I to and I Ks in a voltage and concentration dependent way and narrowed the repolarizing differences among three layers. The analysis of gating kinetics showed ALL accelerated the channel activation of I to in M layers and partly inhibit the channel openings of I to in Epi, M and Endo cells. On the other hand, ALL mainly slowed channel deactivation of I Ks channel in Epi and Endo layers without affecting its activation. Our study gives partially explanation about the mechanisms of transmural inhibition of I to and I Ks channels by ALL in rabbit myocardium. These findings provide novel perspective regarding the anti-arrhythmogenesis application of ALL in clinical settings.

Control method for peak power delivery with limited DC-bus voltage

DOEpatents

Edwards, John; Xu, Longya; Bhargava, Brij B.

2006-09-05

A method for driving a neutral point-clamped multi-level voltage source inverter supplying a synchronous motor is provided. A DC current is received at a neutral point-clamped multi-level voltage source inverter. The inverter has first, second, and third output nodes. The inverter also has a plurality of switches. A desired speed of a synchronous motor connected to the inverter by the first second and third nodes is received by the inverter. The synchronous motor has a rotor and the speed of the motor is defined by the rotational rate of the rotor. A position of the rotor is sensed, current flowing to the motor out of at least two of the first, second, and third output nodes is sensed, and predetermined switches are automatically activated by the inverter responsive to the sensed rotor position, the sensed current, and the desired speed.

Intracellular recording of action potentials by nanopillar electroporation.

PubMed

Xie, Chong; Lin, Ziliang; Hanson, Lindsey; Cui, Yi; Cui, Bianxiao

2012-02-12

Action potentials have a central role in the nervous system and in many cellular processes, notably those involving ion channels. The accurate measurement of action potentials requires efficient coupling between the cell membrane and the measuring electrodes. Intracellular recording methods such as patch clamping involve measuring the voltage or current across the cell membrane by accessing the cell interior with an electrode, allowing both the amplitude and shape of the action potentials to be recorded faithfully with high signal-to-noise ratios. However, the invasive nature of intracellular methods usually limits the recording time to a few hours, and their complexity makes it difficult to simultaneously record more than a few cells. Extracellular recording methods, such as multielectrode arrays and multitransistor arrays, are non-invasive and allow long-term and multiplexed measurements. However, extracellular recording sacrifices the one-to-one correspondence between the cells and electrodes, and also suffers from significantly reduced signal strength and quality. Extracellular techniques are not, therefore, able to record action potentials with the accuracy needed to explore the properties of ion channels. As a result, the pharmacological screening of ion-channel drugs is usually performed by low-throughput intracellular recording methods. The use of nanowire transistors, nanotube-coupled transistors and micro gold-spine and related electrodes can significantly improve the signal strength of recorded action potentials. Here, we show that vertical nanopillar electrodes can record both the extracellular and intracellular action potentials of cultured cardiomyocytes over a long period of time with excellent signal strength and quality. Moreover, it is possible to repeatedly switch between extracellular and intracellular recording by nanoscale electroporation and resealing processes. Furthermore, vertical nanopillar electrodes can detect subtle changes in action potentials induced by drugs that target ion channels.

Intracellular recording of action potentials by nanopillar electroporation

NASA Astrophysics Data System (ADS)

Xie, Chong; Lin, Ziliang; Hanson, Lindsey; Cui, Yi; Cui, Bianxiao

2012-03-01

Action potentials have a central role in the nervous system and in many cellular processes, notably those involving ion channels. The accurate measurement of action potentials requires efficient coupling between the cell membrane and the measuring electrodes. Intracellular recording methods such as patch clamping involve measuring the voltage or current across the cell membrane by accessing the cell interior with an electrode, allowing both the amplitude and shape of the action potentials to be recorded faithfully with high signal-to-noise ratios. However, the invasive nature of intracellular methods usually limits the recording time to a few hours, and their complexity makes it difficult to simultaneously record more than a few cells. Extracellular recording methods, such as multielectrode arrays and multitransistor arrays, are non-invasive and allow long-term and multiplexed measurements. However, extracellular recording sacrifices the one-to-one correspondence between the cells and electrodes, and also suffers from significantly reduced signal strength and quality. Extracellular techniques are not, therefore, able to record action potentials with the accuracy needed to explore the properties of ion channels. As a result, the pharmacological screening of ion-channel drugs is usually performed by low-throughput intracellular recording methods. The use of nanowire transistors, nanotube-coupled transistors and micro gold-spine and related electrodes can significantly improve the signal strength of recorded action potentials. Here, we show that vertical nanopillar electrodes can record both the extracellular and intracellular action potentials of cultured cardiomyocytes over a long period of time with excellent signal strength and quality. Moreover, it is possible to repeatedly switch between extracellular and intracellular recording by nanoscale electroporation and resealing processes. Furthermore, vertical nanopillar electrodes can detect subtle changes in action potentials induced by drugs that target ion channels.

Effect of hypobaric hypoxia on the P2X receptors of pyramidal cells in the immature rat hippocampus CA1 sub-field.

PubMed

Zhao, Yan-Dong; Cheng, Sai-Yu; Ou, Shan; Xiao, Zhi; He, Wen-Juan; Jian-Cui; Ruan, Huai-Zhen

2012-01-01

This study was designed to evaluate the effect of hypobaric hypoxia (HH) on the function and expression of P2X receptors in rat hippocampus CA1 pyramidal cells. The functional changes of P2X receptors were investigated through the cell HH model and the expressional alterations of P2X receptors were observed through the animal HH model. P2X receptors mediated currents were recorded from the freshly dissociated CA1 pyramidal cells of 7-day-old SD rats by whole cell patch clamp recording. The expression and distribution of P2X receptors were observed through immunohistochemistry and western blot at HH 3-day and 7-day. In acute HH conditions, the amplitudes of ATP evoked peak currents were decreased compared to control. The immunohistochemistry and western blot results reflected there was no change in P2X receptors expression after 3 days HH injury, while P2X receptors expression was up-regulated in response to 7 days HH injury. These findings supported the possibility that the function of P2X receptors was sensitive to HH damage and long-term function decrease should result in the expression increase of P2X receptors.

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

The properties of single cones isolated from the tiger salamander retina

PubMed Central

Attwell, David; Werblin, Frank S.; Wilson, Martin

1982-01-01

1. The properties of isolated single cones were studied using the voltage-clamp technique, with two micro-electrodes inserted under visual control. 2. Single cones had input resistances, when impaled with two electrodes, of up to 270 MΩ. This is probably lower than the true membrane resistance, because of damage by the impaling electrodes. The cone capacitance was about 85 pF. 3. The cone membrane contains a time-dependent current, IB, controlled by voltage, and a separate photosensitive current. 4. The gated current, IB, is an inward current with a reversal potential around -25 mV. It is activated by hyperpolarization over the range -30 to -80 mV, and at constant voltage obeys first order (exponential) kinetics. The gating time constant is typically 50 ms at the resting potential of -45 mV, rises to 170 ms at -70 mV, and decreases for further hyperpolarization. 5. The spectral sensitivity curve of the cone light response peaks at 620 nm wave-length, and is narrower than the nomogram for vitamin A2-based pigments. The light responses of isolated cones are spectrally univariant. 6. Voltage-clamped photocurrents were recorded at various membrane potentials, for light steps of various intensities. The photocurrent reversed at around -8 mV. The time course of the photocurrent, for a given intensity, was approximately independent of voltage (although its magnitude was voltage-dependent). The shape of the peak current—voltage relation of the light-sensitive current was independent of light intensity (although its magnitude was intensity-dependent). 7. These results can be explained if: (a) light simply changes the number of photosensitive channels open, without altering the properties of an open channel; (b) the reactions controlling the production of internal transmitter, the binding of internal transmitter to the photosensitive channels, and the closing and opening of the channels are unaffected by the electric field in the cone membrane, even though at least some of these reactions take place in the membrane. 8. IB plays only a small role in shaping the cone voltage response to light. ImagesPlate 1 PMID:7131315

Glutamate receptor activation in the kindled dentate gyrus.

PubMed

Behr, J; Heinemann, U; Mody, I

2000-01-01

The contribution of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA), and kainate receptor activation to the enhanced seizure susceptibility of the dentate gyrus was investigated in an experimental model of temporal lobe epilepsy. Using the specific NMDA and AMPA receptor antagonists D-APV and SYM 2206, we examined alterations in glutamate receptor-dependent synaptic currents 48 hours and 28 days after kindling in field-potential and voltage-clamp recordings. Forty-eight hours after kindling, the fractions of AMPA and NMDA receptor-mediated excitatory postsynaptic current components shifted dramatically in favor of the NMDA receptor-mediated response. Four weeks after kindling, however, AMPA and NMDA receptor-mediated excitatory postsynaptic currents reverted to control-like values. Neither single nor repetitive perforant path stimuli evoked kainate receptor-mediated excitatory postsynaptic currents in dentate gyrus granule cells of control or kindled rats. The enhanced excitability of the kindled dentate gyrus 48 hours after the last seizure most likely results from transiently enhanced NMDA receptor activation. The NMDA receptor seems to play a critical role in the induction of the kindled state rather than in the persistence of the enhanced seizure susceptibility.

Enhancement of delayed-rectifier potassium conductance by low concentrations of local anaesthetics in spinal sensory neurones

PubMed Central

Olschewski, Andrea; Wolff, Matthias; Bräu, Michael E; Hempelmann, Gunter; Vogel, Werner; Safronov, Boris V

2002-01-01

Combining the patch-clamp recordings in slice preparation with the ‘entire soma isolation' method we studied action of several local anaesthetics on delayed-rectifier K+ currents in spinal dorsal horn neurones.Bupivacaine, lidocaine and mepivacaine at low concentrations (1–100 μM) enhanced delayed-rectifier K+ current in intact neurones within the spinal cord slice, while exhibiting a partial blocking effect at higher concentrations (>100 μM). In isolated somata 0.1–10 μM bupivacaine enhanced delayed-rectifier K+ current by shifting its steady-state activation characteristic and the voltage-dependence of the activation time constant to more negative potentials by 10–20 mV.Detailed analysis has revealed that bupivacaine also increased the maximum delayed-rectifier K+ conductance by changing the open probability, rather than the unitary conductance, of the channel.It is concluded that local anaesthetics show a dual effect on delayed-rectifier K+ currents by potentiating them at low concentrations and partially suppressing at high concentrations. The phenomenon observed demonstrated the complex action of local anaesthetics during spinal and epidural anaesthesia, which is not restricted to a suppression of Na+ conductance only. PMID:12055132

Muscarinic receptor-mediated excitation of rat intracardiac ganglion neurons.

PubMed

Hirayama, Michiko; Ogata, Masanori; Kawamata, Tomoyuki; Ishibashi, Hitoshi

2015-08-01

Modulation of the membrane excitability of rat parasympathetic intracardiac ganglion neurons by muscarinic receptors was studied using an amphotericin B-perforated patch-clamp recording configuration. Activation of muscarinic receptors by oxotremorine-M (OxoM) depolarized the membrane, accompanied by repetitive action potentials. OxoM evoked inward currents under voltage-clamp conditions at a holding potential of -60 mV. Removal of extracellular Ca(2+) markedly increased the OxoM-induced current (IOxoM). The inward IOxoM in the absence of extracellular Ca(2+) was fully inhibited by removal of extracellular Na(+), indicating the involvement of non-selective cation channels. The IOxoM was inhibited by organic cation channel antagonists including SKF-96365 and ML-204. The IOxoM was antagonized by muscarinic receptor antagonists with the following potency: 4-DAMP > pirenzepine = darifenacin > methoctramine. Muscarinic toxin 7 (MT-7), a highly selective inhibitor for M1 receptor, produced partial inhibition of the IOxoM. In the presence of MT-7, concentration-inhibition curve of the M3-preferring antagonist darifenacin was shifted to the left. These results suggest the contribution of M1 and M3 receptors to the OxoM response. The IOxoM was inhibited by U-73122, a phospholipase C inhibitor. The membrane-permeable IP3 receptor blocker xestospongin C also inhibited the IOxoM. Furthermore, pretreatment with thapsigargin and BAPTA-AM inhibited the IOxoM, while KN-62, a blocker of Ca(2+)/calmodulin-dependent protein kinase II, had no effect. These results suggest that the activation mechanism involves a PLC pathway, release of Ca(2+) from intracellular Ca(2+) stores and calmodulin. The cation channels activated by muscarinic receptors may play an important role in neuronal membrane depolarization in rat intracardiac ganglion neurons. Copyright © 2015 Elsevier Ltd. All rights reserved.

Zolpidem modulation of phasic and tonic GABA currents in the rat dorsal motor nucleus of the vagus

PubMed Central

Gao, Hong; Smith, Bret N.

2010-01-01

Zolpidem is a widely prescribed sleep aid with relative selectivity for GABAA receptors containing α1–3 subunits. We examined the effects of zolpidem on the inhibitory currents mediated by GABAA receptors using whole-cell patch-clamp recordings from DMV neurons in transverse brainstem slices from rat. Zolpidem prolonged the decay time of mIPSCs and of muscimol-evoked whole-cell GABAergic currents, and it occasionally enhanced the amplitude of mIPSCs. The effects were blocked by flumazenil, a benzodiazepine antagonist. Zolpidem also hyperpolarized the resting membrane potential, with a concomitant decrease in input resistance and action potential firing activity in a subset of cells. Zolpidem did not clearly alter the GABAA receptor-mediated tonic current (Itonic) under baseline conditions, but after elevating extracellular GABA concentration with nipecotic acid, a non-selective GABA transporter blocker, zolpidem consistently and significantly increased the tonic GABA current. This increase was suppressed by flumazenil and gabazine. These results suggest that α1–3 subunits are expressed in synaptic GABAA receptors on DMV neurons. The baseline tonic GABA current is likely not mediated by these same low affinity, zolpidem-sensitive GABAA receptors. However, when the extracellular GABA concentration is increased, zolpidem-sensitive extrasynaptic GABAA receptors containing α1–3 subunits contribute to the Itonic. PMID:20226798

Na/K pump inactivation, subsarcolemmal Na measurements, and cytoplasmic ion turnover kinetics contradict restricted Na spaces in murine cardiac myocytes

PubMed Central

Lu, Fang-Min

2017-01-01

Decades ago, it was proposed that Na transport in cardiac myocytes is modulated by large changes in cytoplasmic Na concentration within restricted subsarcolemmal spaces. Here, we probe this hypothesis for Na/K pumps by generating constitutive transsarcolemmal Na flux with the Na channel opener veratridine in whole-cell patch-clamp recordings. Using 25 mM Na in the patch pipette, pump currents decay strongly during continuous activation by extracellular K (τ, ∼2 s). In contradiction to depletion hypotheses, the decay becomes stronger when pump currents are decreased by hyperpolarization. Na channel currents are nearly unchanged by pump activity in these conditions, and conversely, continuous Na currents up to 0.5 nA in magnitude have negligible effects on pump currents. These outcomes are even more pronounced using 50 mM Li as a cytoplasmic Na congener. Thus, the Na/K pump current decay reflects mostly an inactivation mechanism that immobilizes Na/K pump charge movements, not cytoplasmic Na depletion. When channel currents are increased beyond 1 nA, models with unrestricted subsarcolemmal diffusion accurately predict current decay (τ ∼15 s) and reversal potential shifts observed for Na, Li, and K currents through Na channels opened by veratridine, as well as for Na, K, Cs, Li, and Cl currents recorded in nystatin-permeabilized myocytes. Ion concentrations in the pipette tip (i.e., access conductance) track without appreciable delay the current changes caused by sarcolemmal ion flux. Importantly, cytoplasmic mixing volumes, calculated from current decay kinetics, increase and decrease as expected with osmolarity changes (τ >30 s). Na/K pump current run-down over 20 min reflects a failure of pumps to recover from inactivation. Simulations reveal that pump inactivation coupled with Na-activated recovery enhances the rapidity and effectivity of Na homeostasis in cardiac myocytes. In conclusion, an autoregulatory mechanism enhances cardiac Na/K pump activity when cytoplasmic Na rises and suppresses pump activity when cytoplasmic Na declines. PMID:28606910

Voltage dependence of the rat chorda tympani response to Na+ salts: implications for the functional organization of taste receptor cells.

PubMed

Ye, Q; Heck, G L; DeSimone, J A

1993-07-01

1. Voltage-clamp and current-clamp data were obtained from a circumscribed region of the anterior rat lingual epithelium while simultaneously monitoring the afferent, stimulus-evoked, neural response from the same receptive field. 2. Chorda tympani (CT) responses at constant Na(+)-salt concentration were enhanced by submucosa negative voltage clamp and suppressed by positive voltage clamp. The complete CT response profile, including the time course of adaptation, was not uniquely determined by NaCl concentration alone. The response could be reproduced at different NaCl concentrations by applying a compensating voltage. 3. The form of the concentration and voltage dependence of the CT response indicates that the complete stimulus energy is the Na+ electrochemical potential difference across receptor cell apical membranes, and not Na+ concentration alone. This is the underlying principal behind the equivalence of chemical and electric taste for Na+ salts. 4. CT responses to sodium gluconate (25 and 200 mM) and 25 mM NaCl produced amiloride-insensitive components (AIC) of low magnitude. NaCl at 200 mM produced a significantly larger AIC. The AIC was voltage-clamp independent. The relative magnitude of the AIC was positively correlated with the transepithelial conductance of each salt. This suggests that the large AIC for 200 mM NaCl results from its relatively high permeability through the paracellular pathway. 5. Analysis of the CT response under voltage clamp revealed two anion effects on Na(+)-salt taste, both of which act through the paracellular shunt. 1) Anions modify the transepithelial potential (TP) across tight junctions and thereby modulate the cell receptor potential. This anion effect can be eliminated by voltage clamping the TP. 2) Sufficiently mobile anions facilitate electroneutral diffusion of Na+ salts through tight junctions. This effect is observed especially when Cl- is the anion and when the stimulus concentration favors NaCl influx, allowing Na+ to stimulate receptor cells from the submucosal side. Because the submucosal intercellular spaces are nearly isopotential regions, this effect is insensitive to voltage clamp of the TP. The large AIC associated with this anion effect is due to the low permeability of amiloride.

Nonlinear effects of hyperpolarizing shifts in activation of mutant Nav1.7 channels on resting membrane potential

PubMed Central

Estacion, Mark

2017-01-01

The Nav1.7 sodium channel is preferentially expressed within dorsal root ganglion (DRG) and sympathetic ganglion neurons. Gain-of-function mutations that cause the painful disorder inherited erythromelalgia (IEM) shift channel activation in a hyperpolarizing direction. When expressed within DRG neurons, these mutations produce a depolarization of resting membrane potential (RMP). The biophysical basis for the depolarized RMP has to date not been established. To explore the effect on RMP of the shift in activation associated with a prototypical IEM mutation (L858H), we used dynamic-clamp models that represent graded shifts that fractionate the effect of the mutation on activation voltage dependence. Dynamic-clamp recording from DRG neurons using a before-and-after protocol for each cell made it possible, even in the presence of cell-to-cell variation in starting RMP, to assess the effects of these graded mutant models. Our results demonstrate a nonlinear, progressively larger effect on RMP as the shift in activation voltage dependence becomes more hyperpolarized. The observed differences in RMP were predicted by the “late” current of each mutant model. Since the depolarization of RMP imposed by IEM mutant channels is known, in itself, to produce hyperexcitability of DRG neurons, the development of pharmacological agents that normalize or partially normalize activation voltage dependence of IEM mutant channels merits further study. NEW & NOTEWORTHY Inherited erythromelalgia (IEM), the first human pain disorder linked to a sodium channel, is widely regarded as a genetic model of neuropathic pain. IEM is produced by Nav1.7 mutations that hyperpolarize activation. These mutations produce a depolarization of resting membrane potential (RMP) in dorsal root ganglion neurons. Using dynamic clamp to explore the effect on RMP of the shift in activation, we demonstrate a nonlinear effect on RMP as the shift in activation voltage dependence becomes more hyperpolarized. PMID:28148645

Alcohol interactions with channel activation and desensitization at 5-HT[sub 3] and GABA[sub A] receptors

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

Lovinger, D.M.; Zhou, O.

1992-01-01

Ethanol (EtOH) and trichloroethanol (TCEt) potentiate 5-HT[sub 3] receptor-mediated ion current in NCB-20 neuroblastoma cells and nodose ganglion neurons. TCEt potentiates GABA[sub A] receptor-mediated current in dorsal root ganglion neurons. Whole-cell patch-clamp recording was used to examine the interactions of alcohols with current activation and receptor desensitization. Alcohols increased the potency of 5-HT, consistent with an increase in channel activation rate. Current decay rate increased in the presence of alcohols such that potentiation decreased with time following in onset of agonist + alcohol treatment. Potentiation of 5-HT-activated current by EtOH was 61 [plus minus] 17% above control at the startmore » of application but was absent 10 sec after current onset. Agonist pretreatment decreased potentiation by subsequent agonist + alcohol application. Potentiation by TCEt of 5-HT-activated current decreased from 96% above control with simultaneous application of 5-HT + TCEt to 44% after a 30 sec 5-HT treatment. This agonist- and time-dependent loss of potentiation was observed prior to the onset of current decay when low agonist concentrations were used. Agonist pretreatment appears to drive the channel into an alcohol-insensitive. Current activated by GABA + TCEt recovers from desensitization produced by GABA alone more slowly than recovery tested in the absence of TCEt.« less

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

A fast BK-type KCa current acts as a postsynaptic modulator of temporal selectivity for communication signals.

PubMed

Kohashi, Tsunehiko; Carlson, Bruce A

2014-01-01

Temporal patterns of spiking often convey behaviorally relevant information. Various synaptic mechanisms and intrinsic membrane properties can influence neuronal selectivity to temporal patterns of input. However, little is known about how synaptic mechanisms and intrinsic properties together determine the temporal selectivity of neuronal output. We tackled this question by recording from midbrain electrosensory neurons in mormyrid fish, in which the processing of temporal intervals between communication signals can be studied in a reduced in vitro preparation. Mormyrids communicate by varying interpulse intervals (IPIs) between electric pulses. Within the midbrain posterior exterolateral nucleus (ELp), the temporal patterns of afferent spike trains are filtered to establish single-neuron IPI tuning. We performed whole-cell recording from ELp neurons in a whole-brain preparation and examined the relationship between intrinsic excitability and IPI tuning. We found that spike frequency adaptation of ELp neurons was highly variable. Postsynaptic potentials (PSPs) of strongly adapting (phasic) neurons were more sharply tuned to IPIs than weakly adapting (tonic) neurons. Further, the synaptic filtering of IPIs by tonic neurons was more faithfully converted into variation in spiking output, particularly at short IPIs. Pharmacological manipulation under current- and voltage-clamp revealed that tonic firing is mediated by a fast, large-conductance Ca(2+)-activated K(+) (KCa) current (BK) that speeds up action potential repolarization. These results suggest that BK currents can shape the temporal filtering of sensory inputs by modifying both synaptic responses and PSP-to-spike conversion. Slow SK-type KCa currents have previously been implicated in temporal processing. Thus, both fast and slow KCa currents can fine-tune temporal selectivity.

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.

Role of ion channels and subcellular Ca2+ signaling in arachidonic acid-induced dilation of pressurized retinal arterioles.

PubMed

Kur, Joanna; McGahon, Mary K; Fernández, Jose A; Scholfield, C Norman; McGeown, J Graham; Curtis, Tim M

2014-05-02

To investigate the mechanisms responsible for the dilatation of rat retinal arterioles in response to arachidonic acid (AA). Changes in the diameter of isolated, pressurized rat retinal arterioles were measured in the presence of AA alone and following pre-incubation with pharmacologic agents inhibiting Ca(2+) sparks and oscillations and K(+) channels. Subcellular Ca(2+) signals were recorded in arteriolar myocytes using Fluo-4-based confocal imaging. The effects of AA on membrane currents of retinal arteriolar myocytes were studied using whole-cell perforated patch clamp recording. Arachidonic acid dilated pressurized retinal arterioles under conditions of myogenic tone. Eicosatetraynoic acid (ETYA) exerted a similar effect, but unlike AA, its effects were rapidly reversible. Arachidonic acid-induced dilation was associated with an inhibition of subcellular Ca(2+) signals. Interventions known to block Ca(2+) sparks and oscillations in retinal arterioles caused dilatation and inhibited AA-induced vasodilator responses. Arachidonic acid accelerated the rate of inactivation of the A-type Kv current and the voltage dependence of inactivation was shifted to more negative membrane potentials. It also enhanced voltage-activated and spontaneous large-conductance calcium-activated K(+) (BK) currents, but only at positive membrane potentials. Pharmacologic inhibition of A-type Kv and BK currents failed to block AA-induced vasodilator responses. Arachidonic acid suppressed L-type Ca(2+) currents. These results suggest that AA induces retinal arteriolar vasodilation by inhibiting subcellular Ca(2+)-signaling activity in retinal arteriolar myocytes, most likely through a mechanism involving the inhibition of L-type Ca(2+)-channel activity. Arachidonic acid actions on K(+) currents are inconsistent with a model in which K(+) channels contribute to the vasodilator effects of AA.

Rapid Changes of Potassium Concentration at the Outer Surface of Exposed Single Neurons during Membrane Current Flow

PubMed Central

Neher, E.; Lux, H. D.

1973-01-01

K+-sensitive liquid ion-exchanger microelectrodes are shown to be capable of measuring concentration changes which occur on a millisecond time scale. However, some quaternary ammonium ions, such as tetraethylammonium and acetylcholine, are able to block electrode function when present in concentrations as low as 10-4 to 10-3 M. Changes in extracellular potassium concentration caused by spike activity or voltage clamp pulses of exposed single neurons of the snail Helix pomatia may be measured by these electrodes. Quantitative analysis shows that the total amount of excess potassium found in the vicinity of the cell a short time after a clamp pulse, is in relatively good agreement with the amount of potassium carried by the membrane current. PMID:4689624

Origin of terminal voltage variations due to self-mixing in terahertz frequency quantum cascade lasers.

PubMed

Grier, Andrew; Dean, Paul; Valavanis, Alexander; Keeley, James; Kundu, Iman; Cooper, Jonathan D; Agnew, Gary; Taimre, Thomas; Lim, Yah Leng; Bertling, Karl; Rakić, Aleksandar D; Li, Lianhe H; Harrison, Paul; Linfield, Edmund H; Ikonić, Zoran; Davies, A Giles; Indjin, Dragan

2016-09-19

We explain the origin of voltage variations due to self-mixing in a terahertz (THz) frequency quantum cascade laser (QCL) using an extended density matrix (DM) approach. Our DM model allows calculation of both the current-voltage (I-V) and optical power characteristics of the QCL under optical feedback by changing the cavity loss, to which the gain of the active region is clamped. The variation of intra-cavity field strength necessary to achieve gain clamping, and the corresponding change in bias required to maintain a constant current density through the heterostructure is then calculated. Strong enhancement of the self-mixing voltage signal due to non-linearity of the (I-V) characteristics is predicted and confirmed experimentally in an exemplar 2.6 THz bound-to-continuum QCL.

Differential roles of two delayed rectifier potassium currents in regulation of ventricular action potential duration and arrhythmia susceptibility.

PubMed

Devenyi, Ryan A; Ortega, Francis A; Groenendaal, Willemijn; Krogh-Madsen, Trine; Christini, David J; Sobie, Eric A

2017-04-01

Arrhythmias result from disruptions to cardiac electrical activity, although the factors that control cellular action potentials are incompletely understood. We combined mathematical modelling with experiments in heart cells from guinea pigs to determine how cellular electrical activity is regulated. A mismatch between modelling predictions and the experimental results allowed us to construct an improved, more predictive mathematical model. The balance between two particular potassium currents dictates how heart cells respond to perturbations and their susceptibility to arrhythmias. Imbalances of ionic currents can destabilize the cardiac action potential and potentially trigger lethal cardiac arrhythmias. In the present study, we combined mathematical modelling with information-rich dynamic clamp experiments to determine the regulation of action potential morphology in guinea pig ventricular myocytes. Parameter sensitivity analysis was used to predict how changes in ionic currents alter action potential duration, and these were tested experimentally using dynamic clamp, a technique that allows for multiple perturbations to be tested in each cell. Surprisingly, we found that a leading mathematical model, developed with traditional approaches, systematically underestimated experimental responses to dynamic clamp perturbations. We then re-parameterized the model using a genetic algorithm, which allowed us to estimate ionic current levels in each of the cells studied. This unbiased model adjustment consistently predicted an increase in the rapid delayed rectifier K + current and a drastic decrease in the slow delayed rectifier K + current, and this prediction was validated experimentally. Subsequent simulations with the adjusted model generated the clinically relevant prediction that the slow delayed rectifier is better able to stabilize the action potential and suppress pro-arrhythmic events than the rapid delayed rectifier. In summary, iterative coupling of simulations and experiments enabled novel insight into how the balance between cardiac K + currents influences ventricular arrhythmia susceptibility. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

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

Depolarizing Actions of Hydrogen Sulfide on Hypothalamic Paraventricular Nucleus Neurons

PubMed Central

Khademullah, C. Sahara; Ferguson, Alastair V.

2013-01-01

Hydrogen sulfide (H2S) is a novel neurotransmitter that has been shown to influence cardiovascular functions as well and corticotrophin hormone (CRH) secretion. Since the paraventricular nucleus of the hypothalamus (PVN) is a central relay center for autonomic and endocrine functions, we sought to investigate the effects of H2S on the neuronal population of the PVN. Whole cell current clamp recordings were acquired from the PVN neurons and sodium hydrosulfide hydrate (NaHS) was bath applied at various concentrations (0.1, 1, 10, and 50 mM). NaHS (1, 10, and 50 mM) elicited a concentration-response relationship from the majority of recorded neurons, with almost exclusively depolarizing effects following administration. Cells responded and recovered from NaHS administration quickly and the effects were repeatable. Input differences from baseline and during the NaHS-induced depolarization uncovered a biphasic response, implicating both a potassium and non-selective cation conductance. The results from the neuronal population of the PVN shed light on the possible physiological role that H2S has in autonomic and endocrine function. PMID:23691233

A system for applying rapid warming or cooling stimuli to cells during patch clamp recording or ion imaging.

PubMed

Reid, G; Amuzescu, B; Zech, E; Flonta, M L

2001-10-15

We describe a system for superfusing small groups of cells at a precisely controlled and rapidly adjustable local temperature. Before being applied to the cell or cells under study, solutions are heated or cooled in a chamber of small volume ( approximately 150 microl) and large surface area, sandwiched between four small Peltier elements. The current through the Peltier elements is controlled by a microprocessor using a PID (proportional-integral-derivative) feedback algorithm. The chamber can be heated to at least 60 degrees C and cooled to 0 degrees C, changing its temperature at a maximum rate of about 7 degrees C per second; temperature ramps can be followed under feedback control at up to 4 degrees C per second. Temperature commands can be applied from the digital-to-analogue converter of any laboratory interface or generated digitally by the microprocessor. The peak-to-peak noise contributed by the system does not exceed that contributed by a patch pipette, holder and headstage, making it suitable for single channel as well as whole cell recordings.

Adjustments for the display of quantized ion channel dwell times in histograms with logarithmic bins.

PubMed

Stark, J A; Hladky, S B

2000-02-01

Dwell-time histograms are often plotted as part of patch-clamp investigations of ion channel currents. The advantages of plotting these histograms with a logarithmic time axis were demonstrated by, J. Physiol. (Lond.). 378:141-174), Pflügers Arch. 410:530-553), and, Biophys. J. 52:1047-1054). Sigworth and Sine argued that the interpretation of such histograms is simplified if the counts are presented in a manner similar to that of a probability density function. However, when ion channel records are recorded as a discrete time series, the dwell times are quantized. As a result, the mapping of dwell times to logarithmically spaced bins is highly irregular; bins may be empty, and significant irregularities may extend beyond the duration of 100 samples. Using simple approximations based on the nature of the binning process and the transformation rules for probability density functions, we develop adjustments for the display of the counts to compensate for this effect. Tests with simulated data suggest that this procedure provides a faithful representation of the data.

Triple bar, high efficiency mechanical sealer

DOEpatents

Pak, Donald J.; Hawkins, Samantha A.; Young, John E.

2013-03-19

A clamp with a bottom clamp bar that has a planar upper surface is provided. The clamp may also include a top clamp bar connected to the bottom clamp bar, and a pressure distribution bar between the top clamp bar and the bottom clamp bar. The pressure distribution bar may have a planar lower surface in facing relation to the upper surface of the bottom clamp bar. An object is capable of being disposed in a clamping region between the upper surface and the lower surface. The width of the planar lower surface may be less than the width of the upper surface within the clamping region. Also, the pressure distribution bar may be capable of being urged away from the top clamp bar and towards the bottom clamp bar.

Post clamp

NASA Technical Reports Server (NTRS)

Ramsey, John K. (Inventor); Meyn, Erwin H. (Inventor)

1990-01-01

A pair of spaced collars are mounted at right angles on a clamp body by retaining rings which enable the collars to rotate with respect to the clamp body. Mounting posts extend through aligned holes in the collars and clamp body. Each collar can be clamped onto the inserted post while the clamp body remains free to rotate about the post and collar. The clamp body is selectively clamped onto each post.

Taurine activates strychnine-sensitive glycine receptors in neurons of the rat inferior colliculus.

PubMed

Xu, Han; Zhou, Ke-Qing; Huang, Yi-Na; Chen, Lin; Xu, Tian-Le

2004-09-24

Taurine (Tau) is one of the most abundant free amino acids in the mammalian central nervous system. Whether the neurotransmission of the central auditory system is regulated or modulated by Tau is not clear. In the present study, we investigated the electrophysiological and pharmacological properties of Tau-activated currents in acutely dissociated neurons of the rat inferior colliculus (IC) using whole cell patch clamp recordings. At a holding potential of -60 mV and under a condition of chloride equilibrium potential near 0 mV, Tau activated an inward current and its half-maximal activation concentration was equal to 0.37 mM. The measured reversal potential of Tau-activated currents was close to theoretical chloride equilibrium potential. The currents evoked by Tau at both low (1 mM) and high (10 mM) concentrations were almost completely inhibited by strychnine, a glycine receptor antagonist. The Tau-activated current, however, was not affected by bicuculline, a GABA(A) receptor antagonist. Tau at increased concentrations progressively reduced the current response to subsequent glycine application. At saturated concentrations, Tau-activated current and glycine-activated current were mutually cross-desensitized by each other. These findings indicate that Tau activates glycine receptors in neurons of the rat IC and thus may have a functional role in regulating or modulating the neurotransmission of the central auditory system in mammals.

Dopamine alters glutamate receptor desensitization in retinal horizontal cells of the perch (Perca fluviatilis).

PubMed Central

Schmidt, K F; Kruse, M; Hatt, H

1994-01-01

The patch-clamp technique in combination with a fast liquid filament application system was used to study the effect of dopamine on the glutamate receptor desensitization in horizontal cells of the perch (Perca fluviatilis). Kinetics of ligand-gated ion channels in fish horizontal cells are modulated by dopamine. This modulation is presumably mediated by a cAMP-dependent protein phosphorylation. Before incubation with dopamine, the glutamate receptors of horizontal cells activate and desensitize with fast time constants. In the whole-cell recording mode, fast application of the agonists L-glutamate, quisqualate, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid prior to the dopamine incubation gives rise to fast transient currents with peak values of about 200 pA that desensitize within 100 ms. Kainate as agonist produced higher steady-state currents but no transient currents. After incubation of the cells with dopamine for 3 min, the desensitization was significantly reduced and the agonists L-glutamate, quisqualate, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid induced steady-state currents with amplitudes that were similar to the previously observed transient currents. Kainate-induced currents were only slightly affected. Fast desensitizing currents upon fast application of L-glutamate were also recorded from outside-out patches that were excised from horizontal cells before incubation with dopamine. The currents from excised patches desensitized to a steady-state level of about 0.2 of the peak amplitude with time constants of less than 2 ms. When the outside-out patches were excised from cells after dopamine incubation, steady-state currents were enhanced and no transient currents were observed. The results may indicate that the dopamine-dependent modulation of glutamate-induced currents, which is presumably mediated by a protein phosphorylation, is due to an alteration of the desensitization of the glutamate receptors. PMID:7520178

The actions of mdivi-1, an inhibitor of mitochondrial fission, on rapidly activating delayed-rectifier K⁺ current and membrane potential in HL-1 murine atrial cardiomyocytes.

PubMed

So, Edmund Cheung; Hsing, Chung-Hsi; Liang, Chia-Hua; Wu, Sheng-Nan

2012-05-15

Mdivi-1 is an inhibitor of dynamin related protein 1- (drp1)-mediated mitochondrial fission. However, the mechanisms through which this compound interacts directly with ion currents in heart cells remain unknown. In this study, its effects on ion currents and membrane potential in murine HL-1 cardiomyocytes were investigated. In whole-cell recordings, the addition of mdivi-1 decreased the amplitude of tail current (I(tail)) for the rapidly activating delayed-rectifier K⁺ current (I(Kr)) in a concentration-dependent manner with an IC₅₀ value at 11.6 μM, a value that resembles the inhibition requirement for mitochondrial division. It shifted the activation curve of I(tail) to depolarized voltages with no change in the gating charge. However, mdivi-1 did not alter the rate of recovery from current inactivation. In cell-attached configuration, mdivi-1 inside the pipette suppressed the activity of acetylcholine-activated K⁺ channels without modifying the single-channel conductance. Mdivi-1 (30 μM) slightly depressed the peak amplitude of Na⁺ current with no change in the overall current-voltage relationship. Under current-clamp recordings, addition of mdivi-1 resulted in prolongation for the duration of action potentials (APs) and to increase the firing of spontaneous APs in HL-1 cells. Similarly, in pituitary GH₃ cells, mdivi-1 was effective in directly suppressing the amplitude of ether-à-go-go-related gene-mediated K⁺ current. Therefore, the lengthening of AP duration and increased firing of APs caused by mdivi-1 can be primarily explained by its inhibition of these K⁺ channels enriched in heart cells. The observed effects of mdivi-1 on ion currents were direct and not associated with its inhibition of mitochondrial division. Copyright © 2012 Elsevier B.V. All rights reserved.

Molecular dynamics of alamethicin transmembrane channels from open-channel current noise analysis.

PubMed

Mak, D O; Webb, W W

1995-12-01

Conductance noise measurement of the open states of alamethicin transmembrane channels reveals excess noise attributable to cooperative low-frequency molecular dynamics that can generate fluctuations approximately 1 A rms in the effective channel pore radius. Single-channel currents through both persistent and nonpersistent channels with multiple conductance states formed by purified polypeptide alamethicin in artificial phospholipid bilayers isolated onto micropipettes with gigaohm seals were recorded using a voltage-clamp technique with low background noise (rms noise < 3 pA up to 20 kHz). Current noise power spectra between 100 Hz and 20 kHz of each open channel state showed little frequency dependence. Noise from undetected conductance state transitions was insignificant. Johnson and shot noises were evaluated. Current noise caused by electrolyte concentration fluctuation via diffusion was isolated by its dependence on buffer concentration. After removing these contributions, significant current noise remains in all persistent channel states and increases in higher conductance states. In nonpersistent channels, remaining noise occurs primarily in the lowest two states. These fluctuations of channel conductance are attributed to thermal oscillations of the channel molecular conformation and are modeled as a Langevin translational oscillation of alamethicin molecules moving radially from the channel pore, damped mostly by lipid bilayer viscosity.

Inhibitory Effects of Glycyrrhetinic Acid on the Delayed Rectifier Potassium Current in Guinea Pig Ventricular Myocytes and HERG Channel

PubMed Central

Wu, Delin; Jiang, Linqing; Wu, Hongjin; Wang, Shengqi; Zheng, Sidao; Yang, Jiyuan; Liu, Yuna; Ren, Jianxun; Chen, Xianbing

2013-01-01

Background. Licorice has long been used to treat many ailments including cardiovascular disorders in China. Recent studies have shown that the cardiac actions of licorice can be attributed to its active component, glycyrrhetinic acid (GA). However, the mechanism of action remains poorly understood. Aim. The effects of GA on the delayed rectifier potassium current (I K), the rapidly activating (I Kr) and slowly activating (I Ks) components of I K, and the HERG K+ channel expressed in HEK-293 cells were investigated. Materials and Methods. Single ventricular myocytes were isolated from guinea pig myocardium using enzymolysis. The wild type HERG gene was stably expressed in HEK293 cells. Whole-cell patch clamping was used to record I K (I Kr, I Ks) and the HERG K+ current. Results. GA (1, 5, and 10 μM) inhibited I K (I Kr, I Ks) and the HERG K+ current in a concentration-dependent manner. Conclusion. GA significantly inhibited the potassium currents in a dose- and voltage-dependent manner, suggesting that it exerts its antiarrhythmic action through the prolongation of APD and ERP owing to the inhibition of I K (I Kr, I Ks) and HERG K+ channel. PMID:24069049

Isotonic transport by the Na+-glucose cotransporter SGLT1 from humans and rabbit

PubMed Central

Zeuthen, T; Meinild, A-K; Loo, D D F; Wright, E M; Klaerke, D A

2001-01-01

In order to study its role in steady state water transport, the Na+-glucose cotransporter (SGLT1) was expressed in Xenopus laevis oocytes; both the human and the rabbit clones were tested. The transport activity was monitored as a clamp current and the flux of water followed optically as the change in oocyte volume. SGLT1 has two modes of water transport. First, it acts as a molecular water pump: for each 2 Na+ and 1 sugar molecule 264 water molecules were cotransported in the human SGLT1 (hSGLT1), 424 for the rabbit SGLT1 (rSGLT1). Second, it acts as a water channel. The cotransport of water was tightly coupled to the sugar-induced clamp current. Instantaneous changes in clamp current induced by changes in clamp voltage were accompanied by instantaneous changes in the rate of water transport. The cotransported solution was predicted to be hypertonic, and an osmotic gradient built up across the oocyte membrane with continued transport; this resulted in an additional osmotic influx of water. After 5-10 min a steady state was achieved in which the total influx was predicted to be isotonic with the intracellular solution. With the given expression levels, the steady state water transport was divided about equally between cotransport, osmosis across the SGLT1 and osmosis across the native oocyte membrane. Coexpression of AQP1 with the SGLT1 increased the water permeability more than 10-fold and steady state isotonic transport was achieved after less than 2 s of sugar activation. One-third of the water was cotransported, and the remainder was osmotically driven through the AQP1. The data suggest that SGLT1 has three roles in isotonic water transport: it cotransports water directly, it supplies a passive pathway for osmotic water transport, and it generates an osmotic driving force that can be employed by other pathways, for example aquaporins. PMID:11251046

Current umbilical cord clamping practices and attitudes of obstetricians and midwives toward delayed cord clamping in Saudi Arabia.

PubMed

Ibrahim, Nadia O; Sukkarieh, Hatouf H; Bustami, Rami T; Alshammari, Elaf A; Alasmari, Lama Y; Al-Kadri, Hanan M

2017-01-01

In Saudi Arabia, as in many countries, there is usually no clear definition of the timing of umbilical cord clamping (UCC) in the policies and procedures used by hospitals. The World Health Organization (WHO) recommends delayed cord clamping (DCC) ( > 1 minute after birth) as it can significantly improve hemodynamics and long-term neurodevelopment. To investigate current practices of healthcare professionals on the timing of UCC in Saudi Arabia. Cross-sectional survey. Five tertiary hospitals in Riyadh, Saudi Arabia, during May to October 2016. Obstetricians and midwives completed a widely-used questionnaire on UCC practices. Current UCC practices and attitudes of obstetricians and midwives toward DCC. Eighty-two obstetricians and 75 midwives completed the questionnaire for a response rate of 80%. The majority of respondents were aged 30 years or older (81%) and 84% were females. Most respondents were non-Saudi (66%) and had an educational level of bachelor's degree or higher (72%). Only 42% of respondents reported the existence of UCC guidelines in their practice; 38% reported the existence of a set time for UCC when the neonate was term and healthy, and only 32% had a set time for UCC in preterm neonates. While lower levels of agreement were reported among obstetricians and midwives on the benefits of DCC for babies requiring positive pressure ventilation, the majority of respondents (69-71%) thought that DCC was generally good for both term and preterm babies and that its benefits extend beyond the neonatal period. While the majority of obstetricians and midwives that participated in this study had a positive perception toward DCC, this did not translate to their daily practice as most of these professionals reported a lack of existing UCC guidelines in their institutions. Further studies are warranted to confirm these findings. Participant selection by convenience sampling.

Imaging viscoelastic properties of live cells by AFM: power-law rheology on the nanoscale.

PubMed

Hecht, Fabian M; Rheinlaender, Johannes; Schierbaum, Nicolas; Goldmann, Wolfgang H; Fabry, Ben; Schäffer, Tilman E

2015-06-21

We developed force clamp force mapping (FCFM), an atomic force microscopy (AFM) technique for measuring the viscoelastic creep behavior of live cells with sub-micrometer spatial resolution. FCFM combines force-distance curves with an added force clamp phase during tip-sample contact. From the creep behavior measured during the force clamp phase, quantitative viscoelastic sample properties are extracted. We validate FCFM on soft polyacrylamide gels. We find that the creep behavior of living cells conforms to a power-law material model. By recording short (50-60 ms) force clamp measurements in rapid succession, we generate, for the first time, two-dimensional maps of power-law exponent and modulus scaling parameter. Although these maps reveal large spatial variations of both parameters across the cell surface, we obtain robust mean values from the several hundreds of measurements performed on each cell. Measurements on mouse embryonic fibroblasts show that the mean power-law exponents and the mean modulus scaling parameters differ greatly among individual cells, but both parameters are highly correlated: stiffer cells consistently show a smaller power-law exponent. This correlation allows us to distinguish between wild-type cells and cells that lack vinculin, a dominant protein of the focal adhesion complex, even though the mean values of viscoelastic properties between wildtype and knockout cells did not differ significantly. Therefore, FCFM spatially resolves viscoelastic sample properties and can uncover subtle mechanical signatures of proteins in living cells.

Opioid tolerance in periaqueductal gray neurons isolated from mice chronically treated with morphine

PubMed Central

Bagley, Elena E; Chieng, Billy C H; Christie, MacDonald J; Connor, Mark

2005-01-01

The midbrain periaqueductal gray (PAG) is a major site of opioid analgesic action, and a significant site of cellular adaptations to chronic morphine treatment (CMT). We examined μ-opioid receptor (MOP) regulation of voltage-gated calcium channel currents (ICa) and G-protein-activated K channel currents (GIRK) in PAG neurons from CMT mice. Mice were injected s.c. with 300 mg kg−1 of morphine base in a slow release emulsion three times over 5 days, or with emulsion alone (vehicles). This protocol produced significant tolerance to the antinociceptive effects of morphine in a test of thermal nociception. Voltage clamp recordings were made of ICa in acutely isolated PAG neurons and GIRK in PAG slices. The MOP agonist DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin) inhibited ICa in neurons from CMT mice (230 nM) with a similar potency to vehicle (150 nM), but with a reduced maximal effectiveness (37% inhibition in vehicle neurons, 27% in CMT neurons). Inhibition of ICa by the GABAB agonist baclofen was not altered by CMT. Met-enkephalin-activated GIRK currents recorded in PAG slices were significantly smaller in neurons from CMT mice than vehicles, while GIRK currents activated by baclofen were unaltered. These data demonstrate that CMT-induced antinociceptive tolerance is accompanied by homologous reduction in the effectiveness of MOP agonists to inhibit ICa and activate GIRK. Thus, a reduction in MOP number and/or functional coupling to G proteins accompanies the characteristic cellular adaptations to CMT previously described in PAG neurons. PMID:15980868

Opioid tolerance in periaqueductal gray neurons isolated from mice chronically treated with morphine.

PubMed

Bagley, Elena E; Chieng, Billy C H; Christie, MacDonald J; Connor, Mark

2005-09-01

The midbrain periaqueductal gray (PAG) is a major site of opioid analgesic action, and a significant site of cellular adaptations to chronic morphine treatment (CMT). We examined mu-opioid receptor (MOP) regulation of voltage-gated calcium channel currents (I(Ca)) and G-protein-activated K channel currents (GIRK) in PAG neurons from CMT mice. Mice were injected s.c. with 300 mg kg(-1) of morphine base in a slow release emulsion three times over 5 days, or with emulsion alone (vehicles). This protocol produced significant tolerance to the antinociceptive effects of morphine in a test of thermal nociception. Voltage clamp recordings were made of I(Ca) in acutely isolated PAG neurons and GIRK in PAG slices. The MOP agonist DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin) inhibited I(Ca) in neurons from CMT mice (230 nM) with a similar potency to vehicle (150 nM), but with a reduced maximal effectiveness (37% inhibition in vehicle neurons, 27% in CMT neurons). Inhibition of I(Ca) by the GABA(B) agonist baclofen was not altered by CMT. Met-enkephalin-activated GIRK currents recorded in PAG slices were significantly smaller in neurons from CMT mice than vehicles, while GIRK currents activated by baclofen were unaltered. These data demonstrate that CMT-induced antinociceptive tolerance is accompanied by homologous reduction in the effectiveness of MOP agonists to inhibit I(Ca) and activate GIRK. Thus, a reduction in MOP number and/or functional coupling to G proteins accompanies the characteristic cellular adaptations to CMT previously described in PAG neurons.

Electrophysiological Evidence for the Presence of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Mouse Sperm

PubMed Central

Dulce, Figueiras Fierro; José, Acevedo Juan; Pablo, Martínez; Escoffier, Jessica; Sepúlveda, Francisco V.; Enrique, Balderas; Gerardo, Orta; Pablo, Visconti; Alberto, Darszon

2014-01-01

Mammalian sperm must undergo a maturational process, named capacitation, in the female reproductive tract to fertilize the egg. Sperm capacitation is regulated by a cAMP/PKA pathway and involves increases in intracellular Ca2+, pH, Cl−, protein tyrosine phosphorylation, and in mouse and some other mammals a membrane potential hyperpolarization. The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl− channel modulated by cAMP/PKA and ATP, was detected in mammalian sperm and proposed to modulate capacitation. Our whole-cell patch-clamp recordings from testicular mouse sperm now reveal a Cl− selective component to membrane current that is ATP-dependent, stimulated by cAMP, cGMP and genistein (a CFTR agonist, at low concentrations), and inhibited by DPC and CFTRinh-172, two well-known CFTR antagonists. Furthermore, the Cl− current component activated by cAMP and inhibited by CFTRinh-172 is absent in recordings on testicular sperm from mice possessing the CFTR ΔF508 loss-of-function mutation, indicating that CFTR is responsible for this component. A Cl− selective like current component displaying CFTR characteristics was also found in wild type epididymal sperm bearing the cytoplasmatic droplet. Capacitated sperm treated with CFTRinh-172 undergo a shape change, suggesting that CFTR is involved in cell volume regulation. These findings indicate that functional CFTR channels are present in mouse sperm and their biophysical properties are consistent with their proposed participation in capacitation. PMID:22833409

Use of the Satinsky clamp for hilar clamping during robotic partial nephrectomy: indications, technique, and multi-center outcomes.

PubMed

Abdullah, Newaj; Rahbar, Haider; Barod, Ravi; Dalela, Deepansh; Larson, Jeff; Johnson, Michael; Mass, Alon; Zargar, Homayoun; Kaouk, Jihad; Allaf, Mohamad; Bhayani, Sam; Stifelman, Michael; Rogers, Craig

2017-03-01

A Satinsky clamp may be a backup option for hilar clamping during robotic partial nephrectomy (RPN) if there are challenges with application of bulldog clamps, but there are potential safety concerns. We evaluate outcomes of RPN using Satinsky vs. bulldog clamps, and provide tips for safe use of the Satinsky as a backup option. Using a multi-center database, we identified 1073 patients who underwent RPN between 2006 and 2013, and had information available about method of hilar clamping (bulldog clamp vs. Satinsky clamp). Patient baseline characteristics, tumor features, and perioperative outcomes were compared between the Satinsky and bulldog clamp groups. A Satinsky clamp was used for hilar clamping in 94 (8.8 %) RPN cases, and bulldog clamps were used in 979 (91.2 %) cases. The use of a Satinsky clamp was associated with greater operative time (198 vs. 175 min, p < 0.001), estimated blood loss (EBL, 200 vs. 100 ml, p < 0.001), warm ischemia time (WIT, 20 vs. 19 min, p = 0.036), transfusion rate (12.8 vs. 4.8 %, p = 0.001), and hospital stay (3 vs. 2 days, p < 0.001). Tumor characteristics and number of renal vessels were similar between groups. There were six intraoperative complications in the Satinsky clamp group, but none were directly related to the Satinsky clamp. On multivariable analysis, the use of the Satinsky clamp was not associated with increase in intraoperative or Clavien ≥3 postoperative complications, positive surgical margin rate or percentage change in estimated glomerular filtration rate. A Satinsky clamp can be a backup option for hilar clamping during challenging RPN cases, but requires careful technique, and was rarely necessary.

Effect of sterilization on stiffness and dimensional stability of rubber-dam clamps.

PubMed

Giebink, D L; Mathieu, G P; Hondrum, S O

1996-01-01

Simulated clinical conditions were used to test the effect of sterilization on rubber-dam clamp stiffness and dimension. Sixty Hygienic and Ivory W7 clamps were either steam or dry heat sterilized and compared to controls. Stiffness and dimensional change between Ivory clamp groups was significant (p<.0001); the sterilized clamps showed less change than the controls. Hygienic groups showed a significant different between the control and dry heat groups (p<.05); the sterilized clamps showed less change than the controls. The change in stiffness and interjaw width for all Ivory clamps compared to all Hygienic clamps was significant (p<.0001). The Hygienic clamps changes less than the Ivory clamps. The results indicate that steam and dry heat sterilization do not affect retention of rubber-dam clamps.

Properties of the cromakalim-induced potassium conductance in smooth muscle cells isolated from the rabbit portal vein.

PubMed Central

Beech, D. J.; Bolton, T. B.

1989-01-01

1. Single smooth muscle cells were isolated freshly from the rabbit portal vein and membrane currents were recorded by the whole-cell or excised patch configurations of the patch-clamp technique at room temperature. 2. Cromakalim (Ckm, 10 microM) induced a potassium current (ICkm) that showed no pronounced voltage-dependence and had low current noise. 3. This current, ICkm, was inhibited by (in order of potency): phencyclidine greater than quinidine greater than 4-aminopyridine greater than tetraethylammonium ions (TEA). These drugs inhibited the delayed rectifier current, IdK, which is activated by depolarization of the cell, with the same order of potency. 4. Large conductance calcium-activated potassium channels (LKCa) in isolated membrane patches were blocked by (in order of potency) quinidine greater than TEA approximately phencyclidine. 4-Aminopyridine was ineffective. A similar order of potency was found for block of spontaneous transient outward currents thought to represent bursts of openings of LKCa channels. 5. The low current noise of ICkm at positive potentials, and its susceptibility to inhibitors indicated that it was not carried by LKCa channels, and that it may be carried by channels which underlie IdK. It was observed that when ICkm was activated, IdK was reduced. However, in two experiments, ICkm was much more susceptible to glibenclamide than IdK; possible reasons for this are discussed. PMID:2590772

Neurosteroid dehydroepiandrosterone sulphate inhibits persistent sodium currents in rat medial prefrontal cortex via activation of sigma-1 receptors.

PubMed

Cheng, Zheng-Xiang; Lan, Dan-Mei; Wu, Pei-Ying; Zhu, Yan-Hua; Dong, Yi; Ma, Lan; Zheng, Ping

2008-03-01

Dehydroepiandrosterone sulphate is one of the most important neurosteroids. In the present paper, we studied the effect of dehydroepiandrosterone sulphate on persistent sodium currents and its mechanism and functional consequence with whole-cell patch clamp recording method combined with a pharmacological approach in the rat medial prefrontal cortex slices. The results showed that dehydroepiandrosterone sulphate inhibited the amplitude of persistent sodium currents and the inhibitory effect was significant at 0.1 microM, reached maximum at 1 microM and decreased with the increase in the concentrations of above 1 microM. The effect of dehydroepiandrosterone sulphate on persistent sodium currents was canceled by the Gi protein inhibitor and the protein kinase C inhibitor, but not by the protein kinase A inhibitor. The effect of dehydroepiandrosterone sulphate on persistent sodium currents was also canceled by the sigma-1 receptor blockers and the sigma-1 receptor agonist could mimic the effect of dehydroepiandrosterone sulphate. Dehydroepiandrosterone sulphate had no significant influence on neuronal excitability but could significantly inhibit chemical inhibition of mitochondria-evoked increase in persistent sodium currents. These results suggest that dehydroepiandrosterone sulphate inhibits persistent sodium currents via the activation of sigma-1 receptors-Gi protein-protein kinase C-coupled signaling pathway, and the main functional consequence of this effect of DHEAS is presumably to protect neurons under ischemia.

Comparison between gradient-dependent hydraulic conductivities of roots using the root pressure probe: the role of pressure propagations and implications for the relative roles of parallel radial pathways.

PubMed

Bramley, Helen; Turner, Neil C; Turner, David W; Tyerman, Stephen D

2007-07-01

Hydrostatic pressure relaxations with the root pressure probe are commonly used for measuring the hydraulic conductivity (Lp(r)) of roots. We compared the Lp(r) of roots from species with different root hydraulic properties (Lupinus angustifolius L. 'Merrit', Lupinus luteus L. 'Wodjil', Triticum aestivum L. 'Kulin' and Zea mays L. 'Pacific DK 477') using pressure relaxations, a pressure clamp and osmotic gradients to induce water flow across the root. Only the pressure clamp measures water flow under steady-state conditions. Lp(r) determined by pressure relaxations was two- to threefold greater than Lp(r) from pressure clamps and was independent of the direction of water flow. Lp(r) (pressure clamp) was two- to fourfold higher than for Lp(r) (osmotic) for all species except Triticum aestivum where Lp(r) (pressure clamp) and Lp(r) (osmotic) were not significantly different. A novel technique was developed to measure the propagation of pressure through roots to investigate the cause of the differences in Lp(r). Root segments were connected between two pressure probes so that when root pressure (P(r)) was manipulated by one probe, the other probe recorded changes in P(r). Pressure relaxations did not induce the expected kinetics in pressure in the probe at the other end of the root when axial hydraulic conductance, and probe and root capacitances were accounted for. An electric circuit model of the root was constructed that included an additional capacitance in the root loaded by a series of resistances. This accounted for the double exponential kinetics for intact roots in pressure relaxation experiments as well as the reduced response observed with the double probe experiments. Although there were potential errors with all the techniques, we considered that the measurement of Lp(r) using the pressure clamp was the most unambiguous for small pressure changes, and provided that sufficient time was allowed for pressure propagation through the root. The differences in Lp(r) from different methods of measurement have implications for the models describing water transport through roots and the potential role of aquaporins.

[Effects of tranexamic acid combined with temporary drain clamping on postoperative blood loss in total knee arthroplasty].

PubMed

Dong, Yi-Long; Qian, Yue-Nan; Zhong, Xi-Qiang; Shen, Guang-Jie; Cai, Chun-Yuan

2017-04-25

To evaluate the efficacy and safety of one dose tranexamic acid combined with temporary drain lamping in primary unilateral total knee arthroplasty. Total 160 patients undergoing unilateral primary total knee arthroplasty between January 2012 and December 2013 were randomly divided into four groups(40 cases in each group):group A (the drain was clamped for 2 hours after the operation and the patients received 20 ml physiological saline), group B(the drain was clamped for 2 hours after the operation and the patients received 10 ml tranexamic acid and 10 ml physiological saline), group C (the drain was clamped for 4 hours after the operation and the patients received 20 ml physiological saline) and group D(the drain was clamped for 4 hours after the operation and the patients received 10 ml tranexamic acid and 10 ml physiological saline). The postoperative hemoglobin level, maximum hemoglobin loss, wound drainage, blood loss, the volume of blood transfusion, the number of patients inquiring blood transfusion, venous thrombo embolism rate, and ecchymosis rate were recorded and compared among the four groups. There was no incision infection, severe hypoxia, and symptomatic pulmonary embolism in these groups. There were significant differences in hemoglobin content one day after operation in each group( F =12.26, P =0.000), in the hemoglobin content 7 days after operation in each group( F =20.74, P =0.000), in postoperative drainage in each group( F =38.71, P =0.000);in the amount of invisible red blood cell loss in each group( F =83.41, P =0.000), and in total red blood cell loss in each group( F =102.68, P =0.000). Color Doppler ultrasound examination found that the total incidence of VTE was 3%(5/160) and there were no significant differences in each group( P =0.892). There were no significant differences in postoperative subcutaneous ecchymosis area>1% incidence( P =0.143). Topical tranexami acid treatment combined with temporary clamping of drain for 4 hours could reduce postoperative blood loss, blood transfusion, and ecchymosis rate without increasing the risk of thromboembolic event after total knee arthroplasty.

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

Phospholipase C-independent effects of 3M3FBS in murine colon.

PubMed

Dwyer, Laura; Kim, Hyun Jin; Koh, Byoung Ho; Koh, Sang Don

2010-02-25

The muscarinic receptor subtype M(3) is coupled to Gq/11 proteins. Muscarinic receptor agonists such as carbachol stimulate these receptors that result in activation of phospholipase C (PLC) which hydrolyzes phosphatidylinositol 4,5-bisphosphate into diacylglycerol and Ins(1,4,5)P(3). This pathway leads to excitation and smooth muscle contraction. In this study the PLC agonist, 2, 4, 6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benezenesulfonamide (m-3M3FBS), was used to investigate whether direct PLC activation mimics carbachol-induced excitation. We examined the effects of m-3M3FBS and 2, 4, 6-trimethyl-N-(ortho-3-trifluoromethyl-phenyl)-benzenesulfonamide (o-3M3FBS), on murine colonic smooth muscle tissue and cells by performing conventional microelectrode recordings, isometric force measurements and patch clamp experiments. Application of m-3M3FBS decreased spontaneous contractility in murine colonic smooth muscle without affecting the resting membrane potential. Patch clamp studies revealed that delayed rectifier K(+) channels were reversibly inhibited by m-3M3FBS and o-3M3FBS. The PLC inhibitor, 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), did not prevent this inhibition by m-3M3FBS. Both m-3M3FBS and o-3M3FBS decreased two components of delayed rectifier K(+) currents in the presence of tetraethylammonium chloride or 4-aminopyridine. Ca(2+) currents were significantly suppressed by m-3M3FBS and o-3M3FBS with a simultaneous increase in intracellular Ca(2+). Pretreatment with U73122 did not prevent the decrease in Ca(2+) currents upon m-3M3FBS application. In conclusion, both m-3M3FBS and o-3M3FBS inhibit inward and outward currents via mechanisms independent of PLC acting in an antagonistic manner. In contrast, both compounds also caused an increase in [Ca(2+)](i) in an agonistic manner. Therefore caution must be employed when interpreting their effects at the tissue and cellular level.

Glucose-responsive neurons in the subfornical organ of the rat--a novel site for direct CNS monitoring of circulating glucose.

PubMed

Medeiros, N; Dai, L; Ferguson, A V

2012-01-10

Glucose-sensitive neurons have been identified in a number of CNS regions including metabolic control centers of the hypothalamus. The location of these regions behind the blood-brain barrier restricts them to sensing central, but not circulating glucose concentrations. In this study, we have used patch-clamp electrophysiology to examine whether neurons in a specialized region lacking the blood-brain barrier, the subfornical organ (SFO), are also glucose sensitive. In dissociated SFO neurons, altering the bath concentration of glucose (1 mM, 5 mM, 10 mM) influenced the excitability of 49% of neurons tested (n=67). Glucose-inhibited (GI) neurons depolarized in response to decreased glucose (n=10; mean, 4.6±1.0 mV) or hyperpolarized in response to increased glucose (n=8; mean,-4.4±0.8 mV). In contrast, glucose-excited (GE) neurons depolarized in response to increased glucose (n=9; mean, 6.4±0.4 mV) or hyperpolarized in response to decreased glucose (n=6; mean,-4.8±0.6 mV). Using voltage-clamp recordings, we also identified GI (outward current to increased glucose) and GE (inward current to increased glucose) SFO neurons. The mean glucose-induced inward current had a reversal potential of -24±12 mV (n=5), while GE responses were maintained during sodium-dependent glucose transporter inhibition, supporting the conclusion that GE properties result from the activation of a nonselective cation conductance (NSCC). The glucose-induced outward current had a mean reversal potential of -78±1.2 mV (n=5), while GI responses were not observed in the presence of glibenclamide, suggesting that these properties result from the modulation of K(ATP) channels. These data demonstrate that SFO neurons are glucose responsive, further emphasizing the potential roles of this circumventricular organ as an important sensor and integrator of circulating signals of energy status. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Conservation of cardiac L-type Ca2+ channels and their regulation in Drosophila: A novel genetically-pliable channelopathic model.

PubMed

Limpitikul, Worawan B; Viswanathan, Meera C; O'Rourke, Brian; Yue, David T; Cammarato, Anthony

2018-04-21

Dysregulation of L-type Ca 2+ channels (LTCCs) underlies numerous cardiac pathologies. Understanding their modulation with high fidelity relies on investigating LTCCs in their native environment with intact interacting proteins. Such studies benefit from genetic manipulation of endogenous channels in cardiomyocytes, which often proves cumbersome in mammalian models. Drosophila melanogaster, however, offers a potentially efficient alternative as it possesses a relatively simple heart, is genetically pliable, and expresses well-conserved genes. Fluorescence in situ hybridization confirmed an abundance of Ca-α1D and Ca-α1T mRNA in fly myocardium, which encode subunits that specify hetero-oligomeric channels homologous to mammalian LTCCs and T-type Ca 2+ channels, respectively. Cardiac-specific knockdown of Ca-α1D via interfering RNA abolished cardiac contraction, suggesting Ca-α1D (i.e. A1D) represents the primary functioning Ca 2+ channel in Drosophila hearts. Moreover, we successfully isolated viable single cardiomyocytes and recorded Ca 2+ currents via patch clamping, a feat never before accomplished with the fly model. The profile of Ca 2+ currents recorded in individual cells when Ca 2+ channels were hypomorphic, absent, or under selective LTCC blockage by nifedipine, additionally confirmed the predominance of A1D current across all activation voltages. T-type current, activated at more negative voltages, was also detected. Lastly, A1D channels displayed Ca 2+ -dependent inactivation, a critical negative feedback mechanism of LTCCs, and the current through them was augmented by forskolin, an activator of the protein kinase A pathway. In sum, the Drosophila heart possesses a conserved compendium of Ca 2+ channels, suggesting that the fly may serve as a robust and effective platform for studying cardiac channelopathies. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

Berberine Reduces cAMP-Induced Chloride Secretion in T84 Human Colonic Carcinoma Cells through Inhibition of Basolateral KCNQ1 Channels

PubMed Central

Alzamora, Rodrigo; O’Mahony, Fiona; Ko, Wing-Hung; Yip, Tiffany Wai-Nga; Carter, Derek; Irnaten, Mustapha; Harvey, Brian Joseph

2011-01-01

Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl− secretion in distal colon. The aims of this study were to determine the molecular signaling mechanisms of action of berberine on Cl− secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC50 80 ± 8 μM). In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K+ current by 88%, suggesting inhibition of KCNQ1 K+ channels. Berberine did not affect either apical Cl− conductance or basolateral Na+–K+-ATPase activity. Berberine stimulated p38 MAPK, PKCα and PKA, but had no effect on p42/p44 MAPK and PKCδ. However, berberine pre-treatment prevented stimulation of p42/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl− secretion was partially blocked by HBDDE (∼65%), an inhibitor of PKCα and to a smaller extent by inhibition of p38 MAPK with SB202190 (∼15%). Berberine treatment induced an increase in association between PKCα and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl− secretion through inhibition of basolateral KCNQ1 channels responsible for K+ recycling via a PKCα-dependent pathway. PMID:21747769

Treatment with direct-current stimulation against cingulate seizure-like activity induced by 4-aminopyridine and bicuculline in an in vitro mouse model.

PubMed

Chang, Wei-Pang; Lu, Hsiang-Chin; Shyu, Bai-Chuang

2015-03-01

Clinical studies have shown that cathodal transcranial direct-current stimulation (tDCS) application can produce long-term suppressive effects on drug-resistant seizures. Whether this long-term effect produced by cathodal tDCS can counterbalance the enhancement of synaptic transmission during seizures requires further investigation. Our hypothesis was that the long-term effects of DCS on seizure suppression by the application of cathodal DCS occur through a long-term depression (LTD)-like mechanism. We used a thalamocingulate brain slice preparation combined with a multielectrode array and patch recording to investigate the underlying mechanism of the suppressive effect of DCS on anterior cingulate cortex (ACC) seizures. Patch-clamp recordings showed that cathodal DCS significantly decreased spontaneous excitatory postsynaptic currents (EPSCs) and epileptic EPSCs caused by the 4-aminopyridine. Fifteen minutes of DCS application reliably induced LTD, and the synaptic activation frequency was an important factor in LTD formation. The application of DCS alone without continuous synaptic activation did not induce LTD. Direct-current stimulation-induced LTD appeared to be N-methyl-d-aspartate (NMDA)-dependent, in which the application of the NMDA receptor antagonist D-1-2-amino-5-phosphonopentanoic acid (APV) abolished DCS-induced LTD, and the immediate effect remained. Direct-current stimulation-induced LTD and the long-term effects of DCS on seizure-like activities were also abolished by okadaic acid, a protein phosphatase 1 inhibitor. The long-term effects of DCS on seizures were not influenced by the depotentiation blocker FK-506. Therefore, we conclude that the long-term effects of DCS on seizure-like activities in brain slice occur through an LTD-like mechanism. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

An Approximation to the Adaptive Exponential Integrate-and-Fire Neuron Model Allows Fast and Predictive Fitting to Physiological Data.

PubMed

Hertäg, Loreen; Hass, Joachim; Golovko, Tatiana; Durstewitz, Daniel

2012-01-01

For large-scale network simulations, it is often desirable to have computationally tractable, yet in a defined sense still physiologically valid neuron models. In particular, these models should be able to reproduce physiological measurements, ideally in a predictive sense, and under different input regimes in which neurons may operate in vivo. Here we present an approach to parameter estimation for a simple spiking neuron model mainly based on standard f-I curves obtained from in vitro recordings. Such recordings are routinely obtained in standard protocols and assess a neuron's response under a wide range of mean-input currents. Our fitting procedure makes use of closed-form expressions for the firing rate derived from an approximation to the adaptive exponential integrate-and-fire (AdEx) model. The resulting fitting process is simple and about two orders of magnitude faster compared to methods based on numerical integration of the differential equations. We probe this method on different cell types recorded from rodent prefrontal cortex. After fitting to the f-I current-clamp data, the model cells are tested on completely different sets of recordings obtained by fluctuating ("in vivo-like") input currents. For a wide range of different input regimes, cell types, and cortical layers, the model could predict spike times on these test traces quite accurately within the bounds of physiological reliability, although no information from these distinct test sets was used for model fitting. Further analyses delineated some of the empirical factors constraining model fitting and the model's generalization performance. An even simpler adaptive LIF neuron was also examined in this context. Hence, we have developed a "high-throughput" model fitting procedure which is simple and fast, with good prediction performance, and which relies only on firing rate information and standard physiological data widely and easily available.

High current capacity electrical connector

DOEpatents

Bettis, Edward S.; Watts, Harry L.

1976-01-13

An electrical connector is provided for coupling high current capacity electrical conductors such as copper busses or the like. The connector is arranged in a "sandwiched" configuration in which a conductor plate contacts the busses along major surfaces thereof clamped between two stainless steel backing plates. The conductor plate is provided with a plurality of contact buttons affixed therein in a spaced array such that the caps of the buttons extend above the conductor plate surface to contact the busses. When clamping bolts provided through openings in the sandwiched arrangement are tightened, Belleville springs provided under the rim of each button cap are compressed and resiliently force the caps into contact with the busses' contacting surfaces to maintain a predetermined electrical contact area provided by the button cap tops. The contact area does not change with changing thermal or mechanical stresses applied to the coupled conductors.

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.

Auxiliary resonant DC tank converter

DOEpatents

Peng, Fang Z.

2000-01-01

An auxiliary resonant dc tank (ARDCT) converter is provided for achieving soft-switching in a power converter. An ARDCT circuit is coupled directly across a dc bus to the inverter to generate a resonant dc bus voltage, including upper and lower resonant capacitors connected in series as a resonant leg, first and second dc tank capacitors connected in series as a tank leg, and an auxiliary resonant circuit comprising a series combination of a resonant inductor and a pair of auxiliary switching devices. The ARDCT circuit further includes first clamping means for holding the resonant dc bus voltage to the dc tank voltage of the tank leg, and second clamping means for clamping the resonant dc bus voltage to zero during a resonant period. The ARDCT circuit resonantly brings the dc bus voltage to zero in order to provide a zero-voltage switching opportunity for the inverter, then quickly rebounds the dc bus voltage back to the dc tank voltage after the inverter changes state. The auxiliary switching devices are turned on and off under zero-current conditions. The ARDCT circuit only absorbs ripples of the inverter dc bus current, thus having less current stress. In addition, since the ARDCT circuit is coupled in parallel with the dc power supply and the inverter for merely assisting soft-switching of the inverter without participating in real dc power transmission and power conversion, malfunction and failure of the tank circuit will not affect the functional operation of the inverter; thus a highly reliable converter system is expected.

Electric Fuel Pump Condition Monitor System Using Electricalsignature Analysis

DOEpatents

Haynes, Howard D [Knoxville, TN; Cox, Daryl F [Knoxville, TN; Welch, Donald E [Oak Ridge, TN

2005-09-13

A pump diagnostic system and method comprising current sensing probes clamped on electrical motor leads of a pump for sensing only current signals on incoming motor power, a signal processor having a means for buffering and anti-aliasing current signals into a pump motor current signal, and a computer having a means for analyzing, displaying, and reporting motor current signatures from the motor current signal to determine pump health using integrated motor and pump diagnostic parameters.

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.

Central Nervous System-Toxic Lidocaine Concentrations Unmask L-Type Ca²⁺ Current-Mediated Action Potentials in Rat Thalamocortical Neurons: An In Vitro Mechanism of Action Study.

PubMed

Putrenko, Igor; Ghavanini, Amer A; Meyer Schöniger, Katrin S; Schwarz, Stephan K W

2016-05-01

High systemic lidocaine concentrations exert well-known toxic effects on the central nervous system (CNS), including seizures, coma, and death. The underlying mechanisms are still largely obscure, and the actions of lidocaine on supraspinal neurons have received comparatively little study. We recently found that lidocaine at clinically neurotoxic concentrations increases excitability mediated by Na-independent, high-threshold (HT) action potential spikes in rat thalamocortical neurons. Our goal in this study was to characterize these spikes and test the hypothesis that they are generated by HT Ca currents, previously implicated in neurotoxicity. We also sought to identify and isolate the specific underlying subtype of Ca current. We investigated the actions of lidocaine in the CNS-toxic concentration range (100 μM-1 mM) on ventrobasal thalamocortical neurons in rat brain slices in vitro, using whole-cell patch-clamp recordings aided by differential interference contrast infrared videomicroscopy. Drugs were bath applied; action potentials were generated using current clamp protocols, and underlying currents were identified and isolated with ion channel blockers and electrolyte substitution. Lidocaine (100 μM-1 mM) abolished Na-dependent tonic firing in all neurons tested (n = 46). However, in 39 of 46 (85%) neurons, lidocaine unmasked evoked HT action potentials with lower amplitudes and rates of de-/repolarization compared with control. These HT action potentials remained during the application of tetrodotoxin (600 nM), were blocked by Cd (50 μM), and disappeared after superfusion with an extracellular solution deprived of Ca. These features implied that the unmasked potentials were generated by high-voltage-activated Ca channels and not by Na channels. Application of the L-type Ca channel blocker, nifedipine (5 μM), completely blocked the HT potentials, whereas the N-type Ca channel blocker, ω-conotoxin GVIA (1 μM), had little effect. At clinically CNS-toxic concentrations, lidocaine unmasked in thalamocortical neurons evoked HT action potentials mediated by the L-type Ca current while substantially suppressing Na-dependent excitability. On the basis of the known role of an increase in intracellular Ca in the pathogenesis of local anesthetic neurotoxicity, this novel action represents a plausible contributing candidate mechanism for lidocaine's CNS toxicity in vivo.

High Efficiency Automatic-Power-Controlled and Gain-Clamped EDFA for Broadband Passive Optical Networking Systems

NASA Astrophysics Data System (ADS)

Shen, Jyi-Lai; Wei, Shui-Ken; Lin, Chin-Yuan; Iong Li, Ssu; Huang, Chih-Chuan

2010-04-01

The configuration of a simple improved high efficiency automatic-power-controlled and gain-clamped EDFA (APC-GC-EDFA) for broadband passive optical networking systems (BPON) is presented here. In order to compensate the phase and amplitude variation due to the different distance between the optical line terminal (OLT) and optical network units (ONU), the APC-GC-EDFA need to be employed. A single 980 nm laser module is employed as the primary pump. To extend the bandwidth, all C-band ASE is recycled as the secondary pump to enhance the gain efficiency. An electrical feedback circuit is used as a multi-wavelength channel transmitter monitor for the automatic power control to improve the gain-flattened flatness for stable amplification. The experimental results prove that the EDFA system can provide flatter clamped gain in both C-band and L-band configurations. The gain flatness wavelength ranging from 1530 to 1610 nm is within 32.83 ± 0.64 dB, i.e. below 1.95 %. The gains are clamped at 33.85 ± 0.65 dB for the input signal power of -40 dBm to -10 dBm. The range of noise figure is between 6.37 and 6.56, which is slightly lower compared to that of unclamped amplifiers. This will be very useful for measuring the gain flatness of APC-GC-EDFA. Finally, we have also demonstrated the records of the overall simultaneous dynamics measurements for the new system stabilization. The carrier to noise ratio (CNR) is 49.5 to 50.8 dBc which is above the National Television System Committee (NTSC) standard of 43 dBc, and both composite second order (CSO) 69.2 to 71.5 dBc and composite triple beat (CTB) of 69.8 to 72.2 dBc are above 53 dBc. The recorded corresponding rise-time of 1.087 ms indicates that the system does not exhibit any overshoot of gain or ASE variation due to the signal at the beginning of the pulse.

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.

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.

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.

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

In vitro pharmacologic characterization of a cholinergic receptor on outer hair cells.

PubMed

Erostegui, C; Norris, C H; Bobbin, R P

1994-04-01

Acetylcholine (ACh) is the major neurotransmitter released from the efferent fibers in the cochlea onto the outer hair cells (OHCs). The type of ACh receptor on OHCs and the events subsequent to receptor activation are unclear. Therefore we studied the effect of agonists and antagonists of the ACh receptor on isolated OHCs from the guinea pig. OHCs were recorded from in whole cell voltage and current clamp configuration. ACh induced an increase in outward K+ current (IACh) which hyperpolarized the OHCs. No desensitization to ACh application was observed. Cs+ replaced K+ in carrying the IACh. The IACh is Ca(2+)-dependent, time and voltage sensitive, and different from the IKCa induced by depolarization of the membrane potential. When tested at 100 microM, several agonists also induced outward current responses (acetylcholine > suberyldicholine > or = carbachol > DMPP) whereas nicotine, cytisine and muscarine did not. The IACh response to 10 microM ACh was blocked by low concentrations of traditional and non-traditional-nicotinic antagonists (strychnine > curare > bicuculline > alpha-bungarotoxin > thimethaphan) and by higher concentrations of muscarinic antagonists (atropine > 4-DAMP > AF-DX 116 > pirenzepine). Pharmacologically, the ACh receptor on OHCs is nicotinic.

NMDA Receptor Activity in Circulating Red Blood Cells: Methods of Detection.

PubMed

Makhro, Asya; Kaestner, Lars; Bogdanova, Anna

2017-01-01

Abundance and activity of N-methyl-D-aspartate (NMDA) in circulating red blood cells contributes to the maintenance of intracellular Ca 2+ in these cells and, by doing that, controls red cell volume, membrane stability, and O 2 carrying capacity. Detection of the NMDA receptor activity in red blood cells is challenging as the number of its copies is low and shows substantial cell-to-cell heterogeneity. Receptor abundance is reliably assessed using the radiolabeled antagonist ([ 3 H]MK-801) binding technique. Uptake of Ca 2+ following the NMDA receptor activation is detected in cells loaded with Ca 2+ -sensitive fluorescent dye Fluo-4 AM. Both microfluorescence live-cell imaging and flow cytometry may be used for fluorescence intensity detection. Automated patch clamp is currently used for recording of electric currents triggered by the stimulation of the NMDA receptor. These currents are mediated by the Ca 2+ -sensitive K + (Gardos) channels that open upon Ca 2+ uptake via the active NMDA receptor. Furthermore, K + flux through the Gardos channels induced by the NMDA receptor stimulation in red blood cells may be detected using unidirectional K + ( 86 Rb + ) influx.

Voltage-clamp study of the activation currents and fast block to polyspermy in the egg of Xenopus laevis.

PubMed

Glahn, David; Nuccitelli, Richard

2003-04-01

Voltage-clamped mature, jelly-intact Xenopus eggs were used to carefully examine the ionic currents crossing the plasma membrane before, during, and after fertilization. The bulk of the fertilization current was transient, of large amplitude, and reversed at the predicted Cl- reversal potential. However, the large amplitude fertilization current was preceded by a small, step-like increase in holding current. This small increase in holding current is referred to in this paper as Ion to acknowledge its qualitative similarity to the Ion current previously described in the sea urchin. It was observed in both fertilized and artificially activated eggs, and was found to be unaffected by 10 mm tetra-ethyl ammonium (TEA), a concentration found to block K+ currents in Rana pipiens. Current-voltage relationships are presented for the large fertilization potential, and show that the fertilization currents have a marked outward rectification and are voltage sensitive. These properties are in contrast to the total lack of rectification and slight voltage sensitivity seen before or after the fertilization currents. The time required for sperm to fertilize the egg was found to be voltage dependent with a relatively more depolarized voltage requiring a longer time for fertilization to occur. The percentage of eggs blocked with varying potential levels was determined and this information was fitted to a modified Boltzmann equation having a midpoint of -9 mV.

Clostridium perfringens epsilon toxin targets granule cells in the mouse cerebellum and stimulates glutamate release.

PubMed

Lonchamp, Etienne; Dupont, Jean-Luc; Wioland, Laetitia; Courjaret, Raphaël; Mbebi-Liegeois, Corinne; Jover, Emmanuel; Doussau, Frédéric; Popoff, Michel R; Bossu, Jean-Louis; de Barry, Jean; Poulain, Bernard

2010-09-30

Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca(2+) rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons.

Clostridium perfringens Epsilon Toxin Targets Granule Cells in the Mouse Cerebellum and Stimulates Glutamate Release

PubMed Central

Lonchamp, Etienne; Dupont, Jean-Luc; Wioland, Laetitia; Courjaret, Raphaël; Mbebi-Liegeois, Corinne; Jover, Emmanuel; Doussau, Frédéric; Popoff, Michel R.; Bossu, Jean-Louis; de Barry, Jean; Poulain, Bernard

2010-01-01

Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca2+ rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons. PMID:20941361

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.

VEGF attenuated increase of outward delayed-rectifier potassium currents in hippocampal neurons induced by focal ischemia via PI3-K pathway.

PubMed

Wu, K W; Yang, P; Li, S S; Liu, C W; Sun, F Y

2015-07-09

We recently indicated that the vascular endothelial growth factor (VEGF) protects neurons against hypoxic death via enhancement of tyrosine phosphorylation of Kv1.2, an isoform of the delayed-rectifier potassium channels through activation of the phosphatidylinositol 3-kinase (PI3-K) signaling pathway. The present study investigated whether VEGF could attenuate ischemia-induced increase of the potassium currents in the hippocampal pyramidal neurons of rats after ischemic injury. Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) to induce brain ischemia. The whole-cell patch-clamp technique was used to record the potassium currents of hippocampal neurons in brain slices from the ischemically injured brains of the rats 24h after MCAO. We detected that transient MCAO caused a significant increase of voltage-gated potassium currents (Kv) and outward delayed-rectifier potassium currents (IK), but not outward transient potassium currents (IA), in the ipsilateral hippocampus compared with the sham. Moreover, we found that VEGF could acutely, reversibly and voltage-dependently inhibit the ischemia-induced IK increase. This inhibitory effect of VEGF could be completely abolished by wortmannin, an inhibitor of PI3-K. Our data indicate that VEGF attenuates the ischemia-induced increase of IK via activation of the PI3-K signaling pathway. Published by Elsevier Ltd.

Production of low-background CuSn6-bronze for the CRESST dark-matter-search experiment.

PubMed

Majorovits, B; Kader, H; Kraus, H; Lossin, A; Pantic, E; Petricca, F; Proebst, F; Seidel, W

2009-01-01

One of the most intriguing open questions in modern particle physics is the nature of the dark matter in our universe. As hypothetical weakly interacting massive particles (WIMPs) do interact with ordinary matter extremely rarely, their observation requires a very low-background detector environment regarding radioactivity as well as an advanced detector technique that allows for active discrimination of the still present radioactive contaminations. The CRESST experiment uses detectors operating at milli-Kelvin temperature. Energy deposition in the detectors is recorded via the simultaneous measurement of a phonon-mediated signal and scintillation emitted by the CaWO(4) crystal targets. The entire setup is made of carefully selected materials. In this note we report on the development of ultra-pure bronze (CuSn(6)) wire in small quantities for springs and clamps that are currently being used in the CRESST II setup.

Hybrid electro-optical nanosystem for neurons investigation

NASA Astrophysics Data System (ADS)

Miu, Mihaela; Kleps, Irina; Craciunoiu, Florea; Simion, Monica; Bragaru, Adina; Ignat, Teodora

2010-11-01

The scope of this paper is development of a new laboratory-on-a-chip (LOC) device for biomedical studies consisting of a microfluidic system coupled to microelectronic/optical transducers with nanometric features, commonly called biosensors. The proposed device is a hybrid system with sensing element on silicon (Si) chip and microfluidic system on polydimethylsiloxane (PDMS) substrates, taking into accounts their particular advantages. Different types of nanoelectrode arrays, positioned in the reactor, have been investigated as sensitive elements for electrical detection and the recording of neuron extracellular electric activity has been monitorized in parallel with whole-cell patch-clamp membrane current. Moreover, using an additional porosification process the sensing element became efficient for optical detection also. The preliminary test results demonstrate the functionality of the proposed design and also the fabrication technology, the devices bringing advantages in terms enhancement of sensitivity in both optoelectronic detection schemes.

The sigma-1 receptor modulates NMDA receptor synaptic transmission and plasticity via SK channels in rat hippocampus

PubMed Central

Martina, Marzia; Turcotte, Marie-Eve B; Halman, Samantha; Bergeron, Richard

2007-01-01

The sigma receptor (σR), once considered a subtype of the opioid receptor, is now described as a distinct pharmacological entity. Modulation of N-methyl-d-aspartate receptor (NMDAR) functions by σR-1 ligands is well documented; however, its mechanism is not fully understood. Using patch-clamp whole-cell recordings in CA1 pyramidal cells of rat hippocampus and (+)pentazocine, a high-affinity σR-1 agonist, we found that σR-1 activation potentiates NMDAR responses and long-term potentiation (LTP) by preventing a small conductance Ca2+-activated K+ current (SK channels), known to shunt NMDAR responses, to open. Therefore, the block of SK channels and the resulting increased Ca2+ influx through the NMDAR enhances NMDAR responses and LTP. These results emphasize the importance of the σR-1 as postsynaptic regulator of synaptic transmission. PMID:17068104

The sigma-1 receptor modulates NMDA receptor synaptic transmission and plasticity via SK channels in rat hippocampus.

PubMed

Martina, Marzia; Turcotte, Marie-Eve B; Halman, Samantha; Bergeron, Richard

2007-01-01

The sigma receptor (sigmaR), once considered a subtype of the opioid receptor, is now described as a distinct pharmacological entity. Modulation of N-methyl-D-aspartate receptor (NMDAR) functions by sigmaR-1 ligands is well documented; however, its mechanism is not fully understood. Using patch-clamp whole-cell recordings in CA1 pyramidal cells of rat hippocampus and (+)pentazocine, a high-affinity sigmaR-1 agonist, we found that sigmaR-1 activation potentiates NMDAR responses and long-term potentiation (LTP) by preventing a small conductance Ca2+-activated K+ current (SK channels), known to shunt NMDAR responses, to open. Therefore, the block of SK channels and the resulting increased Ca2+ influx through the NMDAR enhances NMDAR responses and LTP. These results emphasize the importance of the sigmaR-1 as postsynaptic regulator of synaptic transmission.

Retinal input to efferent target amacrine cells in the avian retina

PubMed Central

Lindstrom, Sarah H.; Azizi, Nason; Weller, Cynthia; Wilson, Martin

2012-01-01

The bird visual system includes a substantial projection, of unknown function, from a midbrain nucleus to the contralateral retina. Every centrifugal, or efferent, neuron originating in the midbrain nucleus makes synaptic contact with the soma of a single, unique amacrine cell, the target cell (TC). By labeling efferent neurons in the midbrain we have been able to identify their terminals in retinal slices and make patch clamp recordings from TCs. TCs generate Na+ based action potentials triggered by spontaneous EPSPs originating from multiple classes of presynaptic neurons. Exogenously applied glutamate elicited inward currents having the mixed pharmacology of NMDA, kainate and inward rectifying AMPA receptors. Exogenously applied GABA elicited currents entirely suppressed by GABAzine, and therefore mediated by GABAA receptors. Immunohistochemistry showed the vesicular glutamate transporter, vGluT2, to be present in the characteristic synaptic boutons of efferent terminals, whereas the GABA synthetic enzyme, GAD, was present in much smaller processes of intrinsic retinal neurons. Extracellular recording showed that exogenously applied GABA was directly excitatory to TCs and, consistent with this, NKCC, the Cl− transporter often associated with excitatory GABAergic synapses, was identified in TCs by antibody staining. The presence of excitatory retinal input to TCs implies that TCs are not merely slaves to their midbrain input; instead, their output reflects local retinal activity and descending input from the midbrain. PMID:20650017

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons.

PubMed

Urbano, Francisco J; Luster, Brennon R; D'Onofrio, Stasia; Mahaffey, Susan; Garcia-Rill, Edgar

2016-09-14

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the centrolateral/parafascicular; Cl/Pf nucleus). The activation of either the PPN or Cl/Pf nuclei in vivo has been described to induce the arousal of the animal and an increment in gamma band activity in the cortical electroencephalogram (EEG). The cellular mechanisms for the generation of gamma band oscillations in Reticular Activating System (RAS) neurons are the same as those found to generate gamma band oscillations in other brains nuclei. During current-clamp recordings of PPN neurons (from parasagittal slices from 9 - 25 day-old rats), the use of depolarizing square steps rapidly activated voltage-dependent potassium channels that prevented PPN neurons from being depolarized beyond -25 mV. Injecting 1 - 2 sec long depolarizing current ramps gradually depolarized PPN membrane potential resting values towards 0 mV. However, injecting depolarizing square pulses generated gamma-band oscillations of membrane potential that showed to be smaller in amplitude compared to the oscillations generated by ramps. All experiments were performed in the presence of voltage-gated sodium channels and fast synaptic receptors blockers. It has been shown that the activation of high-threshold voltage-dependent calcium channels underlie gamma-band oscillatory activity in PPN neurons. Specific methodological and pharmacological interventions are described here, providing the necessary tools to induce and sustain PPN subthreshold gamma band oscillation in vitro.

Revealing dynamically-organized receptor ion channel clusters in live cells by a correlated electric recording and super-resolution single-molecule imaging approach.

PubMed

Yadav, Rajeev; Lu, H Peter

2018-03-28

The N-methyl-d-aspartate (NMDA) receptor ion-channel is activated by the binding of ligands, along with the application of action potential, important for synaptic transmission and memory functions. Despite substantial knowledge of the structure and function, the gating mechanism of the NMDA receptor ion channel for electric on-off signals is still a topic of debate. We investigate the NMDA receptor partition distribution and the associated channel's open-close electric signal trajectories using a combined approach of correlating single-molecule fluorescence photo-bleaching, single-molecule super-resolution imaging, and single-channel electric patch-clamp recording. Identifying the compositions of NMDA receptors, their spatial organization and distributions over live cell membranes, we observe that NMDA receptors are organized inhomogeneously: nearly half of the receptor proteins are individually dispersed; whereas others exist in heterogeneous clusters of around 50 nm in size as well as co-localized within the diffraction limited imaging area. We demonstrate that inhomogeneous interactions and partitions of the NMDA receptors can be a cause of the heterogeneous gating mechanism of NMDA receptors in living cells. Furthermore, comparing the imaging results with the ion-channel electric current recording, we propose that the clustered NMDA receptors may be responsible for the variation in the current amplitude observed in the on-off two-state ion-channel electric signal trajectories. Our findings shed new light on the fundamental structure-function mechanism of NMDA receptors and present a conceptual advancement of the ion-channel mechanism in living cells.

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

Selective suppression of the slow-inactivating potassium currents by nootropics in molluscan neurons.

PubMed

Bukanova, Julia V; Solntseva, Elena I; Skrebitsky, Vladimir G

2002-09-01

The role of the voltage-gated K+ channels in the effect of some nootropics was investigated. Earlier, the multiple effect of high concentrations of two nootropics, piracetam and its peptide analogue GVS-111 [Seredenin et al. (1995), US Patent No. 5,439,930], on Ca2+ and K+ currents of molluscan neurons was shown [Solntseva et al. (1997), General Pharmacology 29, 85-89]. In the present work, we describe the selective effect of low concentrations of these nootropics as well as vinpocetine on certain types of K+ current. The experiments were performed on isolated neurons of the land snail Helix pomatia using a two-microelectrode voltage-clamp method. The inward voltage-gated Ca2+ current (ICa) and three subtypes of the outward voltage-gated K+ current were recorded: Ca2+-dependent K+ current (IK(Ca)), delayed rectifying current (IKD), and fast-inactivating K+ current (IA). It has been found that I Ca was not changed in the presence of 30 microM vinpocetine, 100 microM piracetam or 10 nM GVS-111, while slow-inactivating, TEA-sensitive IK(Ca) and IKD were inhibited (IK(Ca) more strongly than IKD). In contrast, the fast-inactivating, 4-AP-sensitive K+ current (IA) was not diminished by low concentrations of piracetam and GVS-111, while vinpocetine even augmented it. A possible role of slow-inactivating subtypes of the K+ channels in the development of different forms of dementia is discussed.

Effects of nerve growth factor on the action potential duration and repolarizing currents in a rabbit model of myocardial infarction

PubMed Central

Lan, Yun-Feng; Zhang, Jian-Cheng; Gao, Jin-Lao; Wang, Xue-Ping; Fang, Zhou; Fu, Yi-Cheng; Chen, Mei-Yan; Lin, Min; Xue, Qiao; Li, Yang

2013-01-01

Objectives To investigate the effect of nerve growth factor (NGF) on the action potential and potassium currents of non-infarcted myocardium in the myocardial infarcted rabbit model. Methods Rabbits with occlusion of the left anterior descending coronary artery were prepared and allowed to recover for eight weeks (healed myocardial infarction, HMI). During ligation surgery of the left coronary artery, a polyethylene tube was placed near the left stellate ganglion in the subcutis of the neck for the purpose of administering NGF 400 U/d for eight weeks (HMI + NGF group). Cardiomyocytes were isolated from regions of the non-infarcted left ventricular wall and the action potentials and ion currents in these cells were recorded using whole-cell patch clamps. Results Compared with HMI and control cardiomyocytes, significant prolongation of APD50 or APD90 (Action potential duration (APD) measured at 50% and 90% of repolarization) in HMI + NGF cardiomyocytes was found. The results showed that the 4-aminopyridine sensitive transient outward potassium current (Ito), the rapidly activated omponent of delayed rectifier potassium current (IKr), the slowly activated component of delayed rectifier potassium current (IKs), and the L-type calcium current (ICaL) were significantly altered in NGF + HMI cardiomyocytes compared with HMI and control cells. Conclusions Our results suggest that NGF treatment significantly prolongs APD in HMI cardiomyocytes and that a decrease in outward potassium currents and an increase of inward Ca2+ current are likely the underlying mechanism of action. PMID:23610573

Effects of nerve growth factor on the action potential duration and repolarizing currents in a rabbit model of myocardial infarction.

PubMed

Lan, Yun-Feng; Zhang, Jian-Cheng; Gao, Jin-Lao; Wang, Xue-Ping; Fang, Zhou; Fu, Yi-Cheng; Chen, Mei-Yan; Lin, Min; Xue, Qiao; Li, Yang

2013-03-01

To investigate the effect of nerve growth factor (NGF) on the action potential and potassium currents of non-infarcted myocardium in the myocardial infarcted rabbit model. Rabbits with occlusion of the left anterior descending coronary artery were prepared and allowed to recover for eight weeks (healed myocardial infarction, HMI). During ligation surgery of the left coronary artery, a polyethylene tube was placed near the left stellate ganglion in the subcutis of the neck for the purpose of administering NGF 400 U/d for eight weeks (HMI + NGF group). Cardiomyocytes were isolated from regions of the non-infarcted left ventricular wall and the action potentials and ion currents in these cells were recorded using whole-cell patch clamps. Compared with HMI and control cardiomyocytes, significant prolongation of APD50 or APD90 (Action potential duration (APD) measured at 50% and 90% of repolarization) in HMI + NGF cardiomyocytes was found. The results showed that the 4-aminopyridine sensitive transient outward potassium current (I to), the rapidly activated omponent of delayed rectifier potassium current (I Kr), the slowly activated component of delayed rectifier potassium current (I Ks), and the L-type calcium current (I CaL) were significantly altered in NGF + HMI cardiomyocytes compared with HMI and control cells. Our results suggest that NGF treatment significantly prolongs APD in HMI cardiomyocytes and that a decrease in outward potassium currents and an increase of inward Ca(2+) current are likely the underlying mechanism of action.

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.

Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats.

PubMed

Zhang, Man; Zang, Kai-Hong; Luo, Jia-Lie; Leung, Fung-Ping; Huang, Yu; Lin, Cheng-Yuan; Yang, Zhi-Jun; Lu, Ai-Ping; Tang, Xu-Dong; Xu, Hong-Xi; Sung, Joseph Jao-yiu; Bian, Zhao-Xiang

2013-11-15

This study aimed to investigate the effect of magnolol (5,5'-diallyl-2,2'-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca(2+) currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3-100 μM). In the presence of Bay K8644 (100 nM), magnolol (10-100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and Nώ-nitro-L-arginine methyl ester (L-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3-100 μM) inhibited the L-type Ca(2+) currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca(2+) channel activity. Copyright © 2013 Elsevier GmbH. All rights reserved.

Cevimeline enhances the excitability of rat superior salivatory neurons.

PubMed

Ueda, Hirotaka; Mitoh, Yoshihiro; Ichikawa, Hiroyuki; Kobashi, Motoi; Yamashiro, Takashi; Matsuo, Ryuji

2009-01-01

Cevimeline, a therapeutic drug for xerostomia, is an agonist of muscarinic acetylcholine receptors (mAChRs), and directly stimulates the peripheral mAChRs of the salivary glands. Since cevimeline is distributed in the brain after its oral administration, it is possible that it affects the central nervous system. However, it is unknown how cevimeline affects the superior salivatory (SS) neurons, which control submandibular salivation. In the present study, we examined the effects of cevimeline on the SS neurons using the whole-cell patch-clamp technique in brain slices. In Wistar rats (6-10 days), the SS neurons were retrogradely labeled by Texas Red applied to the chorda-lingual nerve. Two days after injection, whole-cell recordings were obtained from the labeled cells, and miniature excitatory postsynaptic currents (mEPSCs) were examined. Cevimeline induced the inward currents dose-dependently and increased the frequency of mEPSCs. Therefore, it is suggested that cevimeline enhances the excitability via post- and presynaptic muscarinic receptors in the rat SS neurons. In conclusion, cevimeline may enhance the excitability of the SS neurons.

Stress controlled pulsed direct current co-sputtered Al{sub 1−x}Sc{sub x}N as piezoelectric phase for micromechanical sensor applications

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

Fichtner, Simon, E-mail: sif@tf.uni-kiel.de; Reimer, Tim; Chemnitz, Steffen

2015-11-01

Scandium alloyed aluminum nitride (Al{sub 1−x}Sc{sub x}N) thin films were fabricated by reactive pulsed direct current co-sputtering of separate scandium and aluminum targets with x ≤ 0.37. A significant improvement of the clamped transversal piezoelectric response to strain e{sub 31,f} from −1.28 C/m{sup 2} to −3.01 C/m{sup 2} was recorded, while dielectric constant and loss angle remain low. Further, the built-in stress level of Al{sub 1−x}Sc{sub x}N was found to be tuneable by varying pressure, Ar/N{sub 2} ratio, and Sc content. The thus resulting enhancement of the expectable signal to noise ratio by a factor of 2.1 and the abilitymore » to control built-in stress make the integration of Al{sub 1−x}Sc{sub x}N as the piezoelectric phase of micro-electro-mechanical system sensor applications highly attractive.« less

Transfer characteristics of the hair cell's afferent synapse

NASA Astrophysics Data System (ADS)

Keen, Erica C.; Hudspeth, A. J.

2006-04-01

The sense of hearing depends on fast, finely graded neurotransmission at the ribbon synapses connecting hair cells to afferent nerve fibers. The processing that occurs at this first chemical synapse in the auditory pathway determines the quality and extent of the information conveyed to the central nervous system. Knowledge of the synapse's input-output function is therefore essential for understanding how auditory stimuli are encoded. To investigate the transfer function at the hair cell's synapse, we developed a preparation of the bullfrog's amphibian papilla. In the portion of this receptor organ representing stimuli of 400-800 Hz, each afferent nerve fiber forms several synaptic terminals onto one to three hair cells. By performing simultaneous voltage-clamp recordings from presynaptic hair cells and postsynaptic afferent fibers, we established that the rate of evoked vesicle release, as determined from the average postsynaptic current, depends linearly on the amplitude of the presynaptic Ca2+ current. This result implies that, for receptor potentials in the physiological range, the hair cell's synapse transmits information with high fidelity. auditory system | exocytosis | glutamate | ribbon synapse | synaptic vesicle

Spontaneous sarcoplasmic reticulum calcium release and extrusion from bovine, not porcine, coronary artery smooth muscle.

PubMed Central

Stehno-Bittel, L; Sturek, M

1992-01-01

1. We tested the hypothesis that the Ca(2+)-loaded sarcoplasmic reticulum (SR) of coronary artery smooth muscle spontaneously releases Ca2+ preferentially toward the sarcolemma to be extruded from the cell without increasing the average free myoplasmic [Ca2+] (Ca(im)) concentration. 2. The SR of bovine cells was Ca(2+)-loaded by depolarization-induced Ca2+ influx. Release (unloading) of Ca2+ from the SR during recovery from depolarization was determined by Fura-2 microfluorometry of Ca(im). The SR Ca2+ unloading was maximal following a long (14 min) recovery from depolarization, as shown by the 66% decrease in the peak caffeine-induced Ca(im) transient compared to the Ca(im) transient after a short (2 min) recovery. No increase in Ca(im) occurred during the long recovery. No unloading of the SR Ca2+ store was noted in porcine cells. 3. Approximately 80% of the outward K+ current in bovine and porcine cells was sensitive to subsarcolemmal Ca2+ (Ca(is)) concentrations. Whole-cell voltage clamp using pipette solutions with Ca2+ concentrations clamped between 0 and 1000 nM with Ca(2+)-EGTA or Ca(2+)-BAPTA buffers showed increasing K+ currents (normalized for cell membrane surface area) as a function of both membrane potential and Ca(is). Clamping of Ca(im) and Ca(is) was verified by the lack of changes in K+ current and Fura-2 ratio in response to Ca2+ influx, Ca(2+)-free external solution, or caffeine-induced Ca2+ release. At +30 to +50 mV the K+ current amplitude showed a similar sensitivity to Ca2+ as Fura-2. These data indicate that in this experimental preparation Ca(2+)-activated K+ current is a valid estimate of Ca(is). 4. Simultaneous Ca(im) and Ca(is) measurements in bovine cells which were not Ca(2+)-clamped (2 x 10(-4) M-EGTA pipette solution) showed that during the long recovery period the K+ current (reflecting Ca(is)) increased 55%, while Ca(im) did not change. 5. In quiescent bovine cells the Ca(is) was higher than Ca(im), while the higher resting Ca(is) gradient was not apparent in porcine cells. 6. The Ca(is) concentration was directly related to the amount of Ca2+ in the SR in bovine, but not porcine cells. Depletion of the SR in bovine cells by caffeine resulted in a 58% decrease in K+ current compared to the resting K+ current. 7. Caffeine-induced Ca2+ release caused an increase in Ca(is) which preceded the increase in Ca(im) by approximately 2 s.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1403820

Autoimmune autonomic ganglionopathy

PubMed Central

Wang, Z.; Low, P.A.; Jordan, J.; Freeman, R.; Gibbons, C.H.; Schroeder, C.; Sandroni, P.; Vernino, S.

2008-01-01

Background Autoimmune autonomic ganglionopathy (AAG) is an acquired immune-mediated form of diffuse autonomic failure. Many patients have serum antibodies that bind to the ganglionic acetylcholine receptors (AChRs) that mediate fast synaptic transmission in autonomic ganglia. Previous clinical studies and observations in animal models suggest that AAG is an antibody-mediated neurologic disorder. Methods Using whole-cell patch clamp techniques, we recorded ganglionic AChR currents in cultured human IMR-32 cells and examined the effects of bath application of IgG derived from patients with AAG. Results IgG from seven patients with AAG all produced a progressive decline in whole-cell ganglionic AChR current, whereas IgG from control subjects had no effect. The effect was abolished at low temperature. Fab antibody fragments had no effect unless a secondary antibody was added concurrently. IgG from one patient also produced a more immediate reduction of ganglionic AChR current. Conclusions The characteristics of antibody-mediated inhibition of ganglionic acetylcholine receptor (AChR) current are consistent with modulation and blocking of the membrane AChR, analogous to the effects of muscle AChR antibodies in myasthenia gravis. Our observations demonstrate that antibodies in patients with autoimmune autonomic ganglionopathy (AAG) cause physiologic changes in ganglionic AChR function and confirm that AAG is an antibody-mediated disorder. PMID:17536048

Intrinsic Plasticity Induced by Group II Metabotropic Glutamate Receptors via Enhancement of High Threshold KV Currents in Sound Localizing Neurons

PubMed Central

Hamlet, William R.; Lu, Yong

2016-01-01

Intrinsic plasticity has emerged as an important mechanism regulating neuronal excitability and output under physiological and pathological conditions. Here, we report a novel form of intrinsic plasticity. Using perforated patch clamp recordings, we examined the modulatory effects of group II metabotropic glutamate receptors (mGluR II) on voltage-gated potassium (KV) currents and the firing properties of neurons in the chicken nucleus laminaris (NL), the first central auditory station where interaural time cues are analyzed for sound localization. We found that activation of mGluR II by synthetic agonists resulted in a selective increase of the high threshold KV currents. More importantly, synaptically released glutamate (with reuptake blocked) also enhanced the high threshold KV currents. The enhancement was frequency-coding region dependent, being more pronounced in low frequency neurons compared to middle and high frequency neurons. The intracellular mechanism involved the Gβγ signaling pathway associated with phospholipase C and protein kinase C. The modulation strengthened membrane outward rectification, sharpened action potentials, and improved the ability of NL neurons to follow high frequency inputs. These data suggest that mGluR II provides a feedforward modulatory mechanism that may regulate temporal processing under the condition of heightened synaptic inputs. PMID:26964678

Suppressive effects of diltiazem and verapamil on delayed rectifier K(+)-channel currents in murine thymocytes.

PubMed

Baba, Asuka; Tachi, Masahiro; Maruyama, Yoshio; Kazama, Itsuro

2015-10-01

Lymphocytes predominantly express delayed rectifier K(+)-channels (Kv1.3) in their plasma membranes, and these channels play crucial roles in the lymphocyte activation and proliferation. Since diltiazem and verapamil, which are highly lipophilic Ca(2+) channel blockers (CCBs), exert relatively stronger immunomodulatory effects than the other types of CCBs, they would affect the Kv1.3-channel currents in lymphocytes. Employing the standard patch-clamp whole-cell recording technique in murine thymocytes, we examined the effects of these drugs on the channel currents and the membrane capacitance. Both diltiazem and verapamil significantly suppressed the peak and the pulse-end currents of the channels, although the effects of verapamil were more marked than those of diltiazem. Both drugs significantly lowered the membrane capacitance, indicating the interactions between the drugs and the plasma membranes. This study demonstrated for the first time that CCBs, such as diltiazem and verapamil, exert inhibitory effects on Kv1.3-channels expressed in lymphocytes. The effects of these drugs may be associated with the mechanisms of immunomodulation by which they decrease the production of inflammatory cytokines. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Quantification of atmospheric emissions and energy metrics from simulated clamp kiln technology in the clay brick industry.

PubMed

Akinshipe, Oladapo; Kornelius, Gerrit

2018-05-01

The quantification of atmospheric emissions from clamp kilns in the clay brick industry has met with limited success globally. The complex configuration of clamp kilns using coal or other carbonaceous fuels and uncertainties regarding kiln combustion conditions, has proven to be a hurdle in measurement of emissions and standardization of process and energy metrics. To enable quantification of these metrics, a model kiln was designed to simulate operating conditions and configuration similar to transverse slice of a typical full-scale clamp kiln, but with lower capacity (20,000 to 35,000 bricks per cycle). Hourly measurements of flue gas at extraction duct were recorded for thirteen firing cycles obtained from various source factories, each lasting 8-14 days, for SO 2 , NO 2 , NO, PM, CO and CO 2 emissions in the extraction stack. A statistical mean efficiency for model kiln emissions capturing and channelling capacity was calculated from sulfur mass balance results of batches that lie within 95% confidence interval of assumed true mean (100%) to give 84.2%. Final emission factors (mean ± standard deviation) were quantified as 22.5 ± 18.8 g/brick for CO, 0.14 ± 0.1 g/brick for NO, 0.0 g/brick for NO 2 , 0.14 ± 0.1 g/brick for NO x , 1.07 ± 0.7 g/brick for SO 2 , 378 ± 223 g/brick for CO 2 ; 0.96 ± 0.5 g/brick for PM 10 ; as well as 1.53 g/brick for hydrocarbons. Energy analysis indicate that a significant reduction of 0.9 MJ/kg (36%) in energy use could be achieved by clamp kiln operators, thereby reducing input costs, and significantly reducing atmospheric emissions. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

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

A new type of single-phase five-level inverter

NASA Astrophysics Data System (ADS)

Xu, Zhi; Li, Shengnan; Qin, Risheng; Zhao, Yanhang

2017-11-01

At present, Neutral Point Clamped (NPC) multilevel inverter is widely applied in new energy field. However, it has some disadvantages including low utilization rate of direct current (DC) voltage source and the unbalance of neutral potential. Therefore, a new single-phase five level inverter is proposed in this paper. It has two stage structure, the former stage is equivalent to three level DC/DC converter, and the back stage uses H bridge to realize inverter. Compared with the original central clamp type inverter, the new five level inverter can improve the utilization of DC voltage, and realize the neutral point potential balance with hysteresis comparator.

Gain-clamped semiconductor optical amplifiers based on compensating light: Theoretical model and performance analysis

NASA Astrophysics Data System (ADS)

Jia, Xin-Hong; Wu, Zheng-Mao; Xia, Guang-Qiong

2006-12-01

It is well known that the gain-clamped semiconductor optical amplifier (GC-SOA) based on lasing effect is subject to transmission rate restriction because of relaxation oscillation. The GC-SOA based on compensating effect between signal light and amplified spontaneous emission by combined SOA and fiber Bragg grating (FBG) can be used to overcome this problem. In this paper, the theoretical model on GC-SOA based on compensating light has been constructed. The numerical simulations demonstrate that good gain and noise figure characteristics can be realized by selecting reasonably the FBG insertion position, the peak reflectivity of FBG and the biasing current of GC-SOA.

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

Zn2+ currents are mediated by calcium-permeable AMPA/Kainate channels in cultured murine hippocampal neurones

PubMed Central

Jia, Yousheng; Jeng, Jade-Ming; Sensi, Stefano L; Weiss, John H

2002-01-01

Permeation of the endogenous cation Zn2+ through calcium-permeable AMPA/kainate receptor-gated (Ca-A/K) channels might subserve pathological and/or physiological signalling roles. Voltage-clamp recording was used to directly assess Zn2+ flux through these channels on cultured murine hippocampal neurones. Ca-A/K channels were present in large numbers only on a minority of neurones (Ca-A/K(+) neurones), many of which were GABAergic. The presence of these channels was assessed in whole-cell or outside-out patch recording as the degree of inward rectification of kainate-activated currents, quantified via a rectification index (RI = G+40/G-60), which ranged from <0.4 (strongly inwardly rectifying) to >2 (outwardly rectifying). The specificity of a low RI as an indication of robust Ca-A/K channel expression was verified by two other techniques, kainate-stimulated cobalt-uptake labelling, and fluorescence imaging of kainate-induced increases in intracellular Ca2+. In addition, the degree of inward rectification of kainate-activated currents correlated strongly with the positive shift of the reversal potential (Vrev) upon switching to a sodium-free, 10 mm Ca2+ buffer. With Zn2+ (3 mm) as the only permeant extracellular cation, kainate-induced inward currents were only observed in neurones that had previously been identified as Ca-A/K(+). A comparison between the Vrev observed with 3 mm Zn2+ and that observed with Ca2+ as the permeant cation revealed a PCa/PZn of ≈1.8. Inward currents recorded in 3 mm Ca2+ were unaffected by the addition of 0.3 mm Zn2+, while microfluorimetrically detected increases in the intracellular concentration of Zn2+ in Ca-A/K(+) neurones upon kainate exposure in the presence of 0.3 mm Zn2+ were only mildly attenuated by the addition of 1.8 mm Ca2+. These results provide direct evidence that Zn2+ can carry currents through Ca-A/K channels, and that there is little interference between Ca2+ and Zn2+ in permeating these channels. PMID:12181280

Pistol-shaped dosimeter charger

DOEpatents

Maples, R.A.

A pistol-shaped charger assembly clamps a cylindrical radiation dosimeter against one edge thereof. A triggerlike lever on the handgrip of the assembly is manually pivoted to actuate a piezoelectric current generator held in the handgrip and thereby charge the dosimeter.

Pistol-shaped dosimeter charger

DOEpatents

Maples, Robert A.

1985-01-01

A pistol-shaped charger assembly clamps a cylindrical radiation dosimeter against one edge thereof. A triggerlike lever on the handgrip of the assembly is manually pivoted to actuate a piezoelectric current generator held in the handgrip and thereby charge the dosimeter.

Mathematical modeling of electrical activity of uterine muscle cells.

PubMed

Rihana, Sandy; Terrien, Jeremy; Germain, Guy; Marque, Catherine

2009-06-01

The uterine electrical activity is an efficient parameter to study the uterine contractility. In order to understand the ionic mechanisms responsible for its generation, we aimed at building a mathematical model of the uterine cell electrical activity based upon the physiological mechanisms. First, based on the voltage clamp experiments found in the literature, we focus on the principal ionic channels and their cognate currents involved in the generation of this electrical activity. Second, we provide the methodology of formulations of uterine ionic currents derived from a wide range of electrophysiological data. The model is validated step by step by comparing simulated voltage-clamp results with the experimental ones. The model reproduces successfully the generation of single spikes or trains of action potentials that fit with the experimental data. It allows analyzing ionic channels implications. Likewise, the calcium-dependent conductance influences significantly the cellular oscillatory behavior.

How the early voltage clamp studies of José del Castillo inform "modern" neuroscience.

PubMed

Zottoli, Steven J

2012-10-01

The description of ionic currents that flow across the membrane of the squid giant axon during an action potential sparked an interest in determining whether there were similar currents in vertebrates. The preparation of choice was the node of Ranvier in single myelinated fibers in frog. José del Castillo spent 3 years on the United States mainland from 1956 to 1959. During that time, he collaborated with Jerome Y. Lettvin and John W. Moore. I discuss how these individuals met one another and some of their scientific discoveries using the voltage clamp to study squid giant axons and frog nodes. Much of this work was conducted at the Marine Biological Laboratory in Woods Hole, MA, and I attempt to convey a sense of the unique scientific "melting pot" that existed at the Marine Biological Laboratory and the broader effect that del Castillo had on "modern" neuroscience.

18β-Glycyrrhetinic acid preferentially blocks late Na current generated by ΔKPQ Nav1.5 channels

PubMed Central

Du, Yi-mei; Xia, Cheng-kun; Zhao, Ning; Dong, Qian; Lei, Ming; Xia, Jia-hong

2012-01-01

Aim: To compare the effects of two stereoisomeric forms of glycyrrhetinic acid on different components of Na+ current, HERG and Kv1.5 channel currents. Methods: Wild-type (WT) and long QT syndrome type 3 (LQT-3) mutant ΔKPQ Nav1.5 channels, as well as HERG and Kv1.5 channels were expressed in Xenopus oocytes. In addition, isolated human atrial myocytes were used. Two-microelectrode voltage-clamp technique was used to record the voltage-activated currents. Results: Superfusion of 18β-glycyrrhetinic acid (18β-GA, 1–100 μmol/L) blocked both the peak current (INa,P) and late current (INa,L) generated by WT and ΔKPQ Nav1.5 channels in a concentration-dependent manner, while 18α-glycyrrhetinic acid (18α-GA) at the same concentrations had no effects. 18β-GA preferentially blocked INa,L (IC50=37.2±14.4 μmol/L) to INa,P (IC50=100.4±11.2 μmol/L) generated by ΔKPQ Nav1.5 channels. In human atrial myocytes, 18β-GA (30 μmol/L) inhibited 47% of INa,P and 87% of INa,L induced by Anemonia sulcata toxin (ATX-II, 30 nmol/L). Superfusion of 18β-GA (100 μmol/L) had no effects on HERG and Kv1.5 channel currents. Conclusion: 18β-GA preferentially blocked the late Na current without affecting HERG and Kv1.5 channels. PMID:22609834

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.

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.

Clamp usable as jig and lifting clamp

DOEpatents

Tsuyama, Yoshizo

1976-01-01

There is provided a clamp which is well suited for use as a lifting clamp for lifting and moving materials of assembly in a shipyard, etc. and as a pulling jig in welding and other operations. The clamp comprises a clamp body including a shackle for engagement with a pulling device and a slot for receiving an article, and a pair of jaws provided on the leg portions of the clamp body on the opposite sides of the slot to grip the article in the slot, one of said jaws consisting of a screw rod and the other jaw consisting of a swivel jaw with a spherical surface, whereby when the article clamped in the slot by the pair of jaws tends to slide in any direction with respect to the clamp body, the article is more positively gripped by the pair of jaws.

Activation of µ-opioid receptors and block of KIR3 potassium channels and NMDA receptor conductance by l- and d-methadone in rat locus coeruleus

PubMed Central

Matsui, Aya; Williams, John T

2010-01-01

BACKGROUND AND PURPOSE Methadone activates opioid receptors to increase a potassium conductance mediated by G-protein-coupled, inwardly rectifying, potassium (KIR3) channels. Methadone also blocks KIR3 channels and N-methyl-D-aspartic acid (NMDA) receptors. However, the concentration dependence and stereospecificity of receptor activation and channel blockade by methadone on single neurons has not been characterized. EXPERIMENTAL APPROACH Intracellular and whole-cell recording were made from locus coeruleus neurons in brain slices and the activation of µ-opioid receptors and blockade of KIR3 and NMDA channels with l- and d-methadone was examined. KEY RESULTS The potency of l-methadone, measured by the amplitude of hyperpolarization was 16.5-fold higher than with d-methadone. A maximum hyperpolarization was caused by both enantiomers (∼30 mV); however, the maximum outward current measured with whole-cell voltage-clamp recording was smaller than the current induced by [Met]5enkephalin. The KIR3 conductance induced by activation of α2-adrenoceptors was decreased with high concentrations of l- and d-methadone (10–30 µM). In addition, methadone blocked the resting inward rectifying conductance (KIR). Both l- and d-methadone blocked the NMDA receptor-dependent current. The block of NMDA receptor-dependent current was voltage-dependent suggesting that methadone acted as a channel blocker. CONCLUSIONS AND IMPLICATIONS Methadone activated µ-opioid receptors at low concentrations in a stereospecific manner. KIR3 and NMDA receptor channel block was not stereospecific and required substantially higher concentrations. The separation in the concentration range suggests that the activation of µ-opioid receptors rather than the channel blocking properties mediate both the therapeutic and toxic actions of methadone. PMID:20659105

Alterations of sodium and potassium channels of RGCs in RCS rat with the development of retinal degeneration.

PubMed

Chen, Zhongshan; Song, Yanping; Yao, Junping; Weng, Chuanhuang; Yin, Zheng Qin

2013-11-01

All know that retinitis pigmentosa (RP) is a group of hereditary retinal degenerative diseases characterized by progressive dysfunction of photoreceptors and associated with progressive cells loss; nevertheless, little is known about how rods and cones loss affects the surviving inner retinal neurons and networks. Retinal ganglion cells (RGCs) process and convey visual information from retina to visual centers in the brain. The healthy various ion channels determine the normal reception and projection of visual signals from RGCs. Previous work on the Royal College of Surgeons (RCS) rat, as a kind of classical RP animal model, indicated that, at late stages of retinal degeneration in RCS rat, RGCs were also morphologically and functionally affected. Here, retrograde labeling for RGCs with Fluorogold was performed to investigate the distribution, density, and morphological changes of RGCs during retinal degeneration. Then, patch clamp recording, western blot, and immunofluorescence staining were performed to study the channels of sodium and potassium properties of RGCs, so as to explore the molecular and proteinic basis for understanding the alterations of RGCs membrane properties and firing functions. We found that the resting membrane potential, input resistance, and capacitance of RGCs changed significantly at the late stage of retinal degeneration. Action potential could not be evoked in a part of RGCs. Inward sodium current and outward potassium current recording showed that sodium current was impaired severely but only slightly in potassium current. Expressions of sodium channel protein were impaired dramatically at the late stage of retinal degeneration. The results suggested that the density of RGCs decreased, process ramification impaired, and sodium ion channel proteins destructed, which led to the impairment of electrophysiological functions of RGCs and eventually resulted in the loss of visual function.

Inhibitory Effect of Vascular Endothelial Growth Factor on the Slowly Activating Delayed Rectifier Potassium Current in Guinea Pig Ventricular Myocytes.

PubMed

Lin, Zhenhao; Xing, Wenlu; Gao, Chuanyu; Wang, Xianpei; Qi, Datun; Dai, Guoyou; Zhao, Wen; Yan, Ganxin

2018-01-26

Vascular endothelial growth factor (VEGF) exerts a number of beneficial effects on ischemic myocardium via its angiogenic properties. However, little is known about whether VEGF has a direct effect on the electrical properties of cardiomyocytes. In the present study, we investigated the effects of different concentrations of VEGF on delayed rectifier potassium currents (I K ) in guinea pig ventricular myocytes and their effects on action potential (AP) parameters. I K and AP were recorded by the whole-cell patch clamp method in ventricular myocytes. Cells were superfused with control solution or solution containing VEGF at different concentrations for 10 minutes before recording. Some ventricular myocytes were pretreated with a phosphatidylinositol 3-kinase inhibitor for 1 hour before the addition of VEGF. We found that VEGF inhibited the slowly activating delayed rectifier potassium current (I K s ) in a concentration-dependent manner (18.13±1.04 versus 12.73±0.34, n=5, P =0.001; 12.73±0.34 versus 9.05±1.20, n=5, P =0.036) and prolonged AP duration (894.5±36.92 versus 746.3±33.71, n=5, P =0.021). Wortmannin, a phosphatidylinositol 3-kinase inhibitor, eliminated these VEGF-induced effects. VEGF had no significant effect on the rapidly activating delayed rectifier potassium current (I K r ), resting membrane potential, AP amplitude, or maximal velocity of depolarization. VEGF inhibited I K s in a concentration-dependent manner through a phosphatidylinositol 3-kinase-mediated signaling pathway, leading to AP prolongation. The results indicate a promising therapeutic potential of VEGF in prevention of ventricular tachyarrhythmias under conditions of high sympathetic activity and ischemia. © 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Cable clamp bolt fixture facilitates assembly in close quarters

NASA Technical Reports Server (NTRS)

Sunderland, G. H.

1967-01-01

Cable clamp bolt holding fixture facilitates forming of electrical cable runs in limited equipment space. The fixture engages the threads of the short clamp bolt through the clamp and maintains tension against clamp tendency to open while the operator installs the nut without difficulty.

The transport systems of Ventricaria ventricosa: hypotonic and hypertonic turgor regulation.

PubMed

Bisson, M A; Beilby, M J

2002-11-01

The time course of hypertonic and hypotonic turgor regulation was studied in Ventricaria (Valonia) using pressure probe and I/V(current-voltage) analysis. Of 11 cells, 9 exhibited hypertonic turgor regulation, ranging from 100% regulation in 150 min to 14% regulation (14% recovery of the decrease in turgor) in 314 min. Some cells began regulating immediately, others took up to 90 min to begin. The resting PD (potential difference) became more positive in most cells. The I/V characteristics became more nonlinear with high resistance between -150 and -20 mV and negative conductance region near -70 mV. Prolonged (16 sec) voltage clamps to negative levels (-100 to -150 mV) showed progressively more rapid current turn-off, but subsequent I/V characteristics were not affected. Clamping to +150 mV, however, abolished the high conductance between -50 and +100 mV to yield a uniform high resistance I/V characteristic, similar to that in high [K+]o. Decreasing illumination from 2.02 micromol sec(-1) m(-2) to 0.5 micromol sec(-1)1 m(-2) had a similar effect. Two out of a total of three cells exhibited hypotonic turgor regulation. Both cells started regulating within minutes and achieved near 50% regulation within 50 min. The PD became more negative. The I/V curves exhibited high resistance between +50 and +150 mV. The characteristics were similar to those in cells exposed to low [K+]o. Prolonged voltage clamps to both negative and positive levels showed slow current increase. Decreased illumination increased the membrane resistance.

Split-tapered joint clamping device

DOEpatents

Olsen, Max J.; Schwartz, Jr., John F.

1988-01-01

This invention relates to a clamping device for removably attaching a tool element to a bracket element wherein a bracket element is disposed in a groove in the tool and a clamping member is disposed in said groove and in engagement with a clamping face of the bracket and a wall of the groove and with the clamping member having pivot means engaging the bracket and about which the clamping member rotates.

Inhibitory actions of synthesised polyamine spider toxins and their analogues on Ca2+-activated Cl- currents recorded from cultured DRG neurones from neonatal rats.

PubMed

Sutton, K G; Stapleton, S R; Scott, R H

1998-07-24

The whole cell variant of the patch clamp technique was used to investigate the actions of polyamine spider toxins and their analogues on high voltage-activated Ca2+ currents and Ca2+-activated Cl- currents (I(Cl(Ca))). The actions of synthesised FTX (putative natural toxin from the American funnel web spider), sFTX-3.3, Orn-FTX-3.3, Lys-FTX-3.3, and argiotoxin-636 on cultured dorsal root ganglion neurones from neonatal rats were investigated. Synthesised FTX (1 microM) inhibited I(Cl(Ca)) but did not inhibit high voltage-activated Ca2+ currents. In contrast, sFTX-3.3 (10 microM) inhibited both high voltage-activated Ca2+ currents and the associated I(Cl(Ca)) in near equal proportions. Argiotoxin-636 (1-10 microM) inhibited I(Cl(Ca)) evoked by Ca2+ entry through voltage-activated channels and by intracellular photorelease of Ca2+ from a caged precursor DM-nitrophen. This data indicates that synthesised FTX and argiotoxin-636 directly inhibit Ca2+-activated Cl- channels. In conclusion, the potency of polyamines as non-selective inhibitors of Ca2+ channels and Ca2+-activated Cl- channels is in part determined by the presence of a terminal arginine and this may involve an interaction between terminal guanidino groups and Ca2+ binding sites.

Mitochondrial uncoupling agents antagonize rotenone actions in rat substantia nigra dopamine neurons.

PubMed

Wu, Yan-Na; Munhall, Adam C; Johnson, Steven W

2011-06-13

Mild uncoupling of oxidative phosphorylation has been reported to reduce generation of reactive oxygen species (ROS) and therefore may be neuroprotective. We reported previously that the mitochondrial poison rotenone enhanced currents evoked by N-methyl-D-aspartate (NMDA) by a ROS-dependent mechanism in rat midbrain dopamine neurons. Thus, rotenone, which produces a model of Parkinson's disease in rodents, may increase the risk of dopamine neuron excitotoxicity. The purpose of this study was to test the hypothesis that oxidative phosphorylation uncoupling agents would antagonize the effect of rotenone on NMDA current. We used patch pipettes to record whole-cell currents under voltage-clamp (-60 mV) in substantia nigra dopamine neurons in slices of rat brain. Rotenone, NMDA and uncoupling agents were added to the brain slice superfusate. Inward currents evoked by NMDA (30 μM) more than doubled in amplitude after slices were superfused for 30 min with 100 nM rotenone. Continuous superfusion with the uncoupling agent carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone (1-3 nM) or 2,4-dinitrophenol (100 nM) significantly antagonized and delayed the ability of rotenone to potentiate NMDA currents. Coenzyme Q₁₀ (1-10 nM), which has been reported to facilitate uncoupling protein activity, also antagonized this action of rotenone. These results suggest that mild uncoupling of oxidative phosphorylation may protect dopamine neurons against injury from mitochondrial poisons such as rotenone. Published by Elsevier B.V.

Bilirubin Modulates Acetylcholine Receptors In Rat Superior Cervical Ganglionic Neurons In a Bidirectional Manner

PubMed Central

Zhang, Chengmi; Wang, Zhenmeng; Dong, Jing; Pan, Ruirui; Qiu, Haibo; Zhang, Jinmin; Zhang, Peng; Zheng, Jijian; Yu, Weifeng

2014-01-01

Autonomic dysfunction as a partial contributing factor to cardiovascular instability in jaundiced patients is often associated with increased serum bilirubin levels. Whether increased serum bilirubin levels could directly inhibit sympathetic ganglion transmission by blocking neuronal nicotinic acetylcholine receptors (nAChRs) remains to be elucidated. Conventional patch-clamp recordings were used to study the effect of bilirubin on nAChRs currents from enzymatically dissociated rat superior cervical ganglia (SCG) neurons. The results showed that low concnetrations (0.5 and 2 μM) of bilirubin enhanced the peak ACh-evoked currents, while high concentrations (3 to 5.5 µM) of bilirubin suppressed the currents with an IC50 of 4 ± 0.5 μM. In addition, bilirubin decreased the extent of desensitization of nAChRs in a concentration-dependent manner. This inhibitory effect of bilirubin on nAChRs channel currents was non-competitive and voltage independent. Bilirubin partly improved the inhibitory effect of forskolin on ACh-induced currents without affecting the action of H-89. These data suggest that the dual effects of enhancement and suppression of bilirubin on nAChR function may be ascribed to the action mechanism of positive allosteric modulation and direct blockade. Thus, suppression of sympathetic ganglionic transmission through postganglionic nAChRs inhibition may partially contribute to the adverse cardiovascular effects in jaundiced patients. PMID:25503810

The antiepileptic medications carbamazepine and phenytoin inhibit native sodium currents in murine osteoblasts.

PubMed

Petty, Sandra J; Milligan, Carol J; Todaro, Marian; Richards, Kay L; Kularathna, Pamuditha K; Pagel, Charles N; French, Chris R; Hill-Yardin, Elisa L; O'Brien, Terence J; Wark, John D; Mackie, Eleanor J; Petrou, Steven

2016-09-01

Fracture risk is a serious comorbidity in epilepsy and may relate to the use of antiepileptic drugs (AEDs). Many AEDs inhibit ion channel function, and the expression of these channels in osteoblasts raises the question of whether altered bone signaling increases bone fragility. We aimed to confirm the expression of voltage-gated sodium (NaV ) channels in mouse osteoblasts, and to investigate the action of carbamazepine and phenytoin on NaV channels. Immunocytochemistry was performed on primary calvarial osteoblasts extracted from neonatal C57BL/6J mice and additional RNA sequencing (RNASeq) was included to confirm expression of NaV . Whole-cell patch-clamp recordings were made to identify the native currents expressed and to assess the actions of carbamazepine (50 μm) or phenytoin (50 μm). NaV expression was demonstrated with immunocytochemistry, RNA sequencing, and functionally, with demonstration of robust tetrodotoxin-sensitive and voltage-activated inward currents. Application of carbamazepine or phenytoin resulted in significant inhibition of current amplitude for carbamazepine (31.6 ± 5.9%, n = 9; p < 0.001), and for phenytoin (35.5 ± 6.9%, n = 7; p < 0.001). Mouse osteoblasts express NaV , and native NaV currents are blocked by carbamazepine and phenytoin, supporting our hypothesis that AEDs can directly influence osteoblast function and potentially affect bone strength. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

OxLDL enhances L-type Ca2+ currents via lysophosphatidylcholine-induced mitochondrial reactive oxygen species (ROS) production.

PubMed

Fearon, Ian M

2006-03-01

To examine the mechanisms underlying oxidised LDL- (oxLDL)-induced alterations in Ca(2+) currents, an effect which underlies altered vascular contractility and cardiac myocyte function. Ca(2+) currents (I(Ca)) were recorded by whole-cell patch-clamp in HEK293 cells expressing L-type Ca(2+) channel alpha(1C) subunits or isolated rat ventricular myocytes. oxLDL (but not native LDL) significantly enhanced recombinant I(Ca), an effect mimicked by 1 microM lysophosphatidylcholine (LPC). LPC failed to enhance I(Ca) either in mitochondrial electron transport chain-depleted rho(0) cells, or in the presence of rotenone (1 microM), or MPP(+) (10 microM). The LPC response was similarly ablated by ascorbate (200 microM) or TROLOX (500 microM) and by the mitochondria-targeted antioxidant, MitoQ (250 nM). In myocytes, enhancement of I(Ca) due to LPC was similarly abrogated with rotenone and MitoQ. These data suggest that LPC enhanced recombinant Ca(2+) currents due to increased mitochondrial ROS production. In support with this, LPC enhanced fluorescence in HEK293 cells and cardiac myocytes loaded with a ROS-sensitive mitochondrial dye, reduced mitotracker red. LPC up-regulates L-type Ca(2+) currents due to altered mitochondrial ROS production, an effect which mediates the response of the native I(Ca) in cardiac myocytes to oxLDL.

Actions and mechanisms of action of novel analogues of sotalol on guinea-pig and rabbit ventricular cells.

PubMed Central

Connors, S. P.; Gill, E. W.; Terrar, D. A.

1992-01-01

1. The actions and mechanisms of action of novel analogues of sotalol which prolong cardiac action potentials were investigated in guinea-pig and rabbit isolated ventricular cells. 2. In guinea-pig and rabbit cells the compounds significantly prolonged action potential duration at 20% and 90% repolarization levels without affecting resting membrane potential. In guinea-pig but not rabbit cells there was an increase in action potential amplitude and in rabbit cells there was no change in the shape or position of the 'notch' in the action potential. 3. Possible mechanisms of action were studied in more detail in the case of compound II (1-(4-methanesulphonamidophenoxy)-3-(N-methyl 3,4 dichlorophenylethylamino)-2-propanol). Prolongation of action potential duration continued to occur in the presence of nisoldipine, and calcium currents recorded under voltage-clamp conditions were not reduced by compound II (1 microM). Action potential prolongation by compound II was also unaffected in the presence of 10 microM tetrodotoxin. 4. Compound II (1 microM) did not influence IK1 assessed from the current during ramp changes in membrane potential (20 mV s-1) over the range -90 to -10 mV. 5. Compound II (1 microM) blocked time-dependent delayed rectifier potassium current (IK) activated by step depolarizations and recorded as an outward tail following repolarization. When a submaximal concentration (50 nM) was applied there was no change in the apparent reversal potential of IK.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1393293

Effects of sildenafil on cardiac repolarization.

PubMed

Chiang, Chern-En; Luk, Hsiang-Ning; Wang, Tsui-Min; Ding, Philip Yu-An

2002-08-01

Sudden death has occasionally been reported in patients taking sildenafil. The objective of this study was to investigate the effect of sildenafil on cardiac repolarization. We used conventional microelectrode recording technique in isolated guinea pig papillary muscles and canine Purkinje fibers, whole-cell patch clamp techniques in guinea pig ventricular myocytes, and in vivo ECG measurements in guinea pigs. Action potential duration at 90% repolarization (APD(90)) was not affected by sildenafil in the therapeutic ranges (< or =1 microM), but shortened by higher concentration (> or =10 microM) in both guinea pig papillary muscles and canine Purkinje fibers. D-Sotalol prolonged APD(90) in the same preparations with concentrations > or =1 microM in a reverse frequency-dependent manner. Co-administration of sildenafil (10 and 30 microM) abolished the APD-prolonging effects of D-sotalol (30 microM) and amiodarone (100 microM). Sildenafil, with concentrations up to 30 microM, had no significant effect on both the rapid (I(Kr)) and the slow (I(Ks)) components of the delayed rectifier potassium currents in guinea pig ventricular myocytes. Sildenafil dose-dependently blocked L-type Ca(2+) current (I(Ca,L)), but had no effect on persistent Na(+) current in guinea pig ventricular myocytes. ECG recordings in intact guinea pigs revealed significant shortening of QTc interval by sildenafil (10 and 30 mg/kg orally). The QT-prolonging effects by D,L-sotalol (50 mg/kg) and amiodarone (100 mg/kg) were abolished by sildenafil (30 mg/kg). Sildenafil does not prolong cardiac repolarization. Instead, in supra-therapeutic concentrations, it accelerates cardiac repolarization, presumably through its blocking effect on I(Ca,L).

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.

Tannic acid activates the Kv7.4 and Kv7.3/7.5 K(+) channels expressed in HEK293 cells and reduces tension in the rat mesenteric arteries.

PubMed

Zhang, Yuanyuan; Chu, Xi; Liu, Ling; Zhang, Nan; Guo, Hui; Yang, Fan; Liu, Zhenyi; Dong, Yongsheng; Bao, Yifan; Zhang, Xuan; Zhang, Jianping

2016-04-01

This study investigated the effect of tannic acid (TA), a plant-derived hydrolyzable polyphenol, on Kv7.4 and Kv7.5 K(+) channels and rat mesenteric artery. Whole-cell patch clamp experiments were used to record the Kv7.4 and Kv7.3/7.5 K(+) currents expressed in HEK293 cells; and the tension changes of mesenteric arteries isolated from rats were recorded using small vessel myography apparatus. Tannic acid increases the Kv7.4 and Kv7.3/7.5 K(+) currents in a concentration-dependent manner (median effective concentration (EC50 ) = 27.3 ± 3.6 μm and EC50 = 23.1 ± 3.9 μm, respectively). In addition, 30 μm TA shifts the G-V curve of Kv7.4 and Kv7.3/7.5 K(+) currents to the left by 14.18 and 25.24 mV, respectively, and prolongs the deactivation time constants by 184.44 and 154.77 ms, respectively. Moreover, TA relaxes the vascular tension of rat mesenteric arteries in a concentration-dependent manner (half inhibitory concentration (IC50 ) = 148.7 ± 13.4 μm). These results confirms the vasodilatory effects of TA on rat mesenteric artery and the activating effects on the Kv7.4 and Kv7.3/7.5 K(+) channels, which may be a mechanism to explain the vasodilatory effect and this mechanism can be used in the research of antihypertension. © 2016 Royal Pharmaceutical Society.

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.

Kv2 Channel Regulation of Action Potential Repolarization and Firing Patterns in Superior Cervical Ganglion Neurons and Hippocampal CA1 Pyramidal Neurons

PubMed Central

Liu, Pin W.

2014-01-01

Kv2 family “delayed-rectifier” potassium channels are widely expressed in mammalian neurons. Kv2 channels activate relatively slowly and their contribution to action potential repolarization under physiological conditions has been unclear. We explored the function of Kv2 channels using a Kv2-selective blocker, Guangxitoxin-1E (GxTX-1E). Using acutely isolated neurons, mixed voltage-clamp and current-clamp experiments were done at 37°C to study the physiological kinetics of channel gating and action potentials. In both rat superior cervical ganglion (SCG) neurons and mouse hippocampal CA1 pyramidal neurons, 100 nm GxTX-1E produced near-saturating block of a component of current typically constituting ∼60–80% of the total delayed-rectifier current. GxTX-1E also reduced A-type potassium current (IA), but much more weakly. In SCG neurons, 100 nm GxTX-1E broadened spikes and voltage clamp experiments using action potential waveforms showed that Kv2 channels carry ∼55% of the total outward current during action potential repolarization despite activating relatively late in the spike. In CA1 neurons, 100 nm GxTX-1E broadened spikes evoked from −70 mV, but not −80 mV, likely reflecting a greater role of Kv2 when other potassium channels were partially inactivated at −70 mV. In both CA1 and SCG neurons, inhibition of Kv2 channels produced dramatic depolarization of interspike voltages during repetitive firing. In CA1 neurons and some SCG neurons, this was associated with increased initial firing frequency. In all neurons, inhibition of Kv2 channels depressed maintained firing because neurons entered depolarization block more readily. Therefore, Kv2 channels can either decrease or increase neuronal excitability depending on the time scale of excitation. PMID:24695716

Lifting clamp positively grips structural shapes

NASA Technical Reports Server (NTRS)

Reinhardt, E. C.

1966-01-01

Welded steel clamps securely grip structural shapes of various sizes for crane operations. The clamp has adjustable clamping jaws and screw-operated internal v-jaws and provides greater safety than hoisting slings presently used. The structural member can be rotated in any manner, angle, or direction without being released by the clamp.

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

The advantage of an alternative substrate over Al/NiP disks

NASA Astrophysics Data System (ADS)

Jiaa, Chi L.; Eltoukhy, Atef

1994-02-01

Compact-size disk drives with high storage densities are in high demand due to the popularity of portable computers and workstations. The contact-start-stop (CSS) endurance performance must improve in order to accomodate the higher number of on/off cycles. In this paper, we looked at 65 mm thin-film canasite substrate disks and evaluated their mechanical performance. We compared them with conventional aluminum NiP-plated disks in surface topography, take-off time with changes of skew angles and radius, CSS, drag test and glide height performance, and clamping effect. In addition, a new post-sputter process aimed at the improvement of take-off and glide as well as CSS performances was investigated and demonstrated for the canasite disks. From the test results, it is indicated that canasite achieved a lower take-off velocity, higher clamping resistance, and better glide height and CSS endurance performance. This study concludes that a new generation disk drive equipped with canasite substrate disks will consume less power from the motor due to faster take-off and lighter weight, achieve higher recording density since the head flies lower, can better withstand damage from sliding friction during the CSS operations, and will be less prone to disk distortion from clamping due to its superior mechanical properties.

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.

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.

Characterisation of rebound depolarisation in mice deep dorsal horn neurons in vitro.

PubMed

Rivera-Arconada, Ivan; Lopez-Garcia, Jose A

2015-09-01

Spinal dorsal horn neurons constitute the first relay for pain processing and participate in the processing of other sensory, motor and autonomic information. At the cellular level, intrinsic excitability is a factor contributing to network function. In turn, excitability is set by the array of ionic conductance expressed by neurons. Here, we set out to characterise rebound depolarisation following hyperpolarisation, a feature frequently described in dorsal horn neurons but never addressed in depth. To this end, an in vitro preparation of the spinal cord from mice pups was used combined with whole-cell recordings in current and voltage clamp modes. Results show the expression of H- and/or T-type currents in a significant proportion of dorsal horn neurons. The expression of these currents determines the presence of rebound behaviour at the end of hyperpolarising pulses. T-type calcium currents were associated to high-amplitude rebounds usually involving high-frequency action potential firing. H-currents were associated to low-amplitude rebounds less prone to elicit firing or firing at lower frequencies. For a large proportion of neurons expressing both currents, the H-current constitutes a mechanism to ensure a faster response after hyperpolarisations, adjusting the latency of the rebound firing. We conclude that rebound depolarisation and firing are intrinsic factors to many dorsal horn neurons that may constitute a mechanism to integrate somatosensory information in the spinal cord, allowing for a rapid switch from inhibited-to-excited states.

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

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.

Regulation of the intracellular free calcium concentration in single rat dorsal root ganglion neurones in vitro.

PubMed Central

Thayer, S A; Miller, R J

1990-01-01

1. Simultaneous whole-cell patch-clamp and Fura-2 microfluorimetric recordings of calcium currents (ICa) and the intracellular free Ca2+ concentration ([Ca2+]i) were made from neurones grown in primary culture from the dorsal root ganglion of the rat. 2. Cells held at -80 mV and depolarized to 0 mV elicited a ICa that resulted in an [Ca2+]i transient which was not significantly buffered during the voltage step and lasted long after the cell had repolarized and the current ceased. The process by which the cell buffered [Ca2+]i back to basal levels could best be described with a single-exponential equation. 3. The membrane potential versus ICa and [Ca2+]i relationship revealed that the peak of the [Ca2+]i transient evoked at a given test potential closely paralleled the magnitude of the ICa suggesting that neither voltage-dependent nor Ca2(+)-induced Ca2+ release from intracellular stores made a significant contribution to the [Ca2+]i transient. 4. When the cell was challenged with Ca2+ loads of different magnitude by varying the duration or potential of the test pulse, [Ca2+]i buffering was more effective for larger Ca2+ loads. The relationship between the integrated ICa and the peak of the [Ca2+]i transient reached an asymptote at large Ca2+ loads indicating that Ca2(+)-dependent processes became more efficient or that low-affinity processes had been recruited. 5. Inhibition of Ca2+ influx with neuropeptide Y demonstrated that inhibition of a large ICa produced minor alterations in the peak of the [Ca2+]i transient, while inhibition of smaller currents produced corresponding decreases in the [Ca2+]i transient. Thus, inhibition of the ICa was reflected by a change in the peak [Ca2+]i only when submaximal Ca2+ loads were applied to the cell, implying that modulation of [Ca2+]i is dependent on the activation state of the cells. 6. Intracellular dialysis with the mitochondrial Ca2+ uptake blocker Ruthenium Red in whole-cell patch-clamp experiments removed the buffering component which was responsible for the more efficient removal of [Ca2+]i observed when large Ca2+ loads were applied to the cell. 7. When cells were superfused with 50 mM-K+, [Ca2+]i transients recorded from the cell soma returned to control levels very slowly. Pharmacological studies indicated that mitochondria were cycling Ca2+ during this sustained elevation in [Ca2+]i. In contrast, [Ca2+]i transients recorded from cell processes returned to basal levels relatively rapidly. 8. Extracellular Na(+)-dependent Ca2+ efflux did not significantly contribute to buffering [Ca2+]i transients in dorsal root ganglion neurone cell bodies.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2213592

Dorsoventral differences in Kv7/M-current and its impact on resonance, temporal summation and excitability in rat hippocampal pyramidal cells

PubMed Central

Hönigsperger, Christoph; Marosi, Máté; Murphy, Ricardo; Storm, Johan F

2015-01-01

Key points Kv7 (KCNQ/M) channels are known to control excitability and generate subthreshold M-resonance in CA1 hippocampal pyramidal cells, but their properties and functions have not previously been compared along the dorsoventral (septotemporal) axis We used whole-cell recordings to compare electrophysiological properties of dorsal and ventral CA1 pyramidal cells in hippocampal slices from 3- to 4-week-old rats Blockade of Kv7/M-channels with 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991) had a stronger impact on electrical properties in dorsal than ventral pyramidal cells, including input resistance, temporal summation, M-resonance, spike threshold, medium after-hyperpolarization, excitability, and spike frequency adaptation. Voltage-clamp recordings revealed a larger amplitude and left-shifted voltage dependence of XE991-sensitive current (IM) in dorsal vs. ventral cells. IM-dependent differences in excitability and resonance may be important for rate and phase coding of CA1 place cells along the dorsoventral axis and may enhance epileptiform activity in ventral pyramidal cells. Abstract In rodent hippocampi, the connections, gene expression and functions differ along the dorsoventral (D–V) axis. CA1 pyramidal cells show increasing excitability along the D–V axis, although the underlying mechanism is not known. In the present study, we investigated how the M-current (IM), caused by Kv7/M (KCNQ) potassium channels, and known to often control neuronal excitability, contributes to D–V differences in intrinsic properties of CA1 pyramidal cells. Using whole-cell patch clamp recordings and the selective Kv7/M blocker 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991) in hippocampal slices from 3- to 4-week-old rats, we found that: (i) IM had a stronger impact on subthreshold electrical properties in dorsal than ventral CA1 pyramidal cells, including input resistance, temporal summation of artificial synaptic potentials, and M-resonance; (ii) IM activated at more negative potentials (left-shifted) and had larger peak amplitude in the dorsal than ventral CA1; and (iii) the initial spike threshold (during ramp depolarizations) was elevated, and the medium after-hyperpolarization and spike frequency adaptation were increased (i.e. excitability was lower) in the dorsal rather than ventral CA1. These differences were abolished or reduced by application of XE991, indicating that they were caused by IM. Thus, it appears that IM has stronger effects in dorsal than in ventral rat CA1 pyramidal cells because of a larger maximal M-conductance and left-shifted activation curve in the dorsal cells. These mechanisms may contribute to D–V differences in the rate and phase coding of position by CA1 place cells, and may also enhance epileptiform activity in ventral CA1. PMID:25656084

Selective Arterial Clamping Versus Hilar Clamping for Minimally Invasive Partial Nephrectomy.

PubMed

Yezdani, Mona; Yu, Sue-Jean; Lee, David I

2016-05-01

Partial nephrectomy has become an accepted treatment of cT1 renal masses as it provides improved long-term renal function compared to radical nephrectomy (Campbell et al. J Urol. 182:1271-9, 2009). Hilar clamping is utilized to help reduce bleeding and improve visibility during tumor resection. However, concern over risk of kidney injury with hilar clamping has led to new techniques to reduce length of warm ischemia time (WIT) during partial nephrectomy. These techniques have progressed over the years starting with early hilar unclamping, controlled hypotension during tumor resection, selective arterial clamping, minimal margin techniques, and off-clamp procedures. Selective arterial clamping has progressed significantly over the years. The main question is what are the exact short- and long-term renal effects from increasing clamp time. Moreover, does it make sense to perform these more time-consuming or more complex procedures if there is no long-term preservation of kidney function? More recent studies have shown no difference in renal function 6 months from surgery when selective arterial clamping or even hilar clamping is employed, although there is short-term improved decline in estimated glomerular filtration rate (eGFR) with selective clamping and off-clamp techniques (Komninos et al. BJU Int. 115:921-8, 2015; Shah et al. 117:293-9, 2015; Kallingal et al. BJU Int. doi: 10.1111/bju.13192, 2015). This paper reviews the progression of total hilar clamping to selective arterial clamping (SAC) and the possible difference its use makes on long-term renal function. SAC may be attempted based on surgeon's decision-making, but may be best used for more complex, larger, more central or hilar tumors and in patients who have renal insufficiency at baseline or a solitary kidney.

Prolonged post-inhibitory rebound firing in the cerebellar nuclei mediated by group I mGluR potentiation of L-type Ca currents

PubMed Central

Zheng, Nan; Raman, Indira M.

2011-01-01

Neurons in the cerebellar nuclei fire at accelerated rates for prolonged periods after trains of synaptic inhibition that interrupt spontaneous firing. Both in vitro and in vivo, however, this prolonged rebound firing is favored by strong stimulation of afferents, suggesting that neurotransmitters other than GABA may contribute to the increased firing rates. Here, we tested whether metabotropic glutamate receptors modulate excitability of nuclear cells in cerebellar slices from mouse. In current clamp, the prolonged rebound firing rate after high-frequency synaptic stimulation was reduced by a variety of group I mGluR antagonists, including CPCCOEt (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester), JNJ16259685 ((3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone)+MPEP, or 3-MATIDA (α-amino-5-carboxy-3-methyl-2-thiopheneacetic acid) +MPEP, as long as both mGluR1 and mGluR5 were blocked. This mGluR-dependent acceleration of firing was reduced but still evident when IPSPs were prevented by GABAA receptor antagonists. In voltage clamp, voltage ramps revealed a non-inactivating, low-voltage-activated, nimodipine-sensitive current that was enhanced by the selective group I mGluR agonist s-DHPG ((S)-3,5-dihydroxyphenylglycine). This putative L-type current also increased when mGluRs were activated by trains of evoked synaptic currents instead of direct application of agonist. In current clamp, blocking L-type Ca channels with the specific blocker nifedipine greatly reduced prolonged post-stimulus firing and occluded the effect of adding group I mGluR antagonists. Thus, potentiation of a low-voltage-activated L-type current by synaptically released glutamate accounted nearly fully for the mGluR-dependent acceleration of firing. Together, these data suggest that prolonged rebound firing in the cerebellar nuclei in vivo is most likely to occur when GABAA and mGluRs are simultaneously activated by concurrent excitation and inhibition. PMID:21753005

Measuring beta-cell function relative to insulin sensitivity in youth: Does the hyperglycemic clamp suffice?

USDA-ARS?s Scientific Manuscript database

To compare beta-cell function relative to insulin sensitivity, disposition index (DI), calculated from two clamps (2cDI, insulin sensitivity from the hyperinsulinemic-euglycemic clamp and first-phase insulin from the hyperglycemic clamp) with the DI calculated from the hyperglycemic clamp alone (hcD...

21 CFR 882.4460 - Neurosurgical head holder (skull clamp).

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Neurosurgical head holder (skull clamp). 882.4460... holder (skull clamp). (a) Identification. A neurosurgical head holder (skull clamp) is a device used to clamp the patient's skull to hold head and neck in a particular position during surgical procedures. (b...

Non-equilibrium voltage noise generated by ion transport through pores.

PubMed

Frehland, E; Solleder, P

1985-01-01

In this paper, we describe a systematic approach to the theoretical analysis of non-equilibrium voltage noise that arises from ions moving through pores in membranes. We assume that an ion must cross one or two barriers in the pore in order to move from one side of the membrane to the other. In our analysis, we consider the following factors: a) surface charge as a variable in the kinetic equations, b) linearization of the kinetic equations, c) master equation approach to fluctuations. To analyze the voltage noise arising from ion movement through a two barrier (i.e., one binding site) pore, we included the effects of ions in the channel's interior on the voltage noise. The current clamp is considered as a white noise generating additional noise in the system. In contrast to what is found for current noise, at low frequencies the voltage noise intensity is reduced by increasing voltage across the membrane. With this approach, we demonstrate explicitly for the examples treated that, apart from additional noise generated by the current clamp, the non-equilibrium voltage fluctuations can be related to the current fluctuations by the complex admittance.

A methodology for achieving high-speed rates for artificial conductance injection in electrically excitable biological cells.

PubMed

Butera, R J; Wilson, C G; Delnegro, C A; Smith, J C

2001-12-01

We present a novel approach to implementing the dynamic-clamp protocol (Sharp et al., 1993), commonly used in neurophysiology and cardiac electrophysiology experiments. Our approach is based on real-time extensions to the Linux operating system. Conventional PC-based approaches have typically utilized single-cycle computational rates of 10 kHz or slower. In thispaper, we demonstrate reliable cycle-to-cycle rates as fast as 50 kHz. Our system, which we call model reference current injection (MRCI); pronounced merci is also capable of episodic logging of internal state variables and interactive manipulation of model parameters. The limiting factor in achieving high speeds was not processor speed or model complexity, but cycle jitter inherent in the CPU/motherboard performance. We demonstrate these high speeds and flexibility with two examples: 1) adding action-potential ionic currents to a mammalian neuron under whole-cell patch-clamp and 2) altering a cell's intrinsic dynamics via MRCI while simultaneously coupling it via artificial synapses to an internal computational model cell. These higher rates greatly extend the applicability of this technique to the study of fast electrophysiological currents such fast a currents and fast excitatory/inhibitory synapses.

Mechanisms of zolpidem-induced long QT syndrome: acute inhibition of recombinant hERG K+ channels and action potential prolongation in human cardiomyocytes derived from induced pluripotent stem cells

PubMed Central

Jehle, J; Ficker, E; Wan, X; Deschenes, I; Kisselbach, J; Wiedmann, F; Staudacher, I; Schmidt, C; Schweizer, PA; Becker, R; Katus, HA; Thomas, D

2013-01-01

Background and Purpose Zolpidem, a short-acting hypnotic drug prescribed to treat insomnia, has been clinically associated with acquired long QT syndrome (LQTS) and torsade de pointes (TdP) tachyarrhythmia. LQTS is primarily attributed to reduction of cardiac human ether-a-go-go-related gene (hERG)/IKr currents. We hypothesized that zolpidem prolongs the cardiac action potential through inhibition of hERG K+ channels. Experimental Approach Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record hERG currents from Xenopus oocytes and from HEK 293 cells. In addition, hERG protein trafficking was evaluated in HEK 293 cells by Western blot analysis, and action potential duration (APD) was assessed in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. Key Results Zolpidem caused acute hERG channel blockade in oocytes (IC50 = 61.5 μM) and in HEK 293 cells (IC50 = 65.5 μM). Mutation of residues Y652 and F656 attenuated hERG inhibition, suggesting drug binding to a receptor site inside the channel pore. Channels were blocked in open and inactivated states in a voltage- and frequency-independent manner. Zolpidem accelerated hERG channel inactivation but did not affect I–V relationships of steady-state activation and inactivation. In contrast to the majority of hERG inhibitors, hERG cell surface trafficking was not impaired by zolpidem. Finally, acute zolpidem exposure resulted in APD prolongation in hiPSC-derived cardiomyocytes. Conclusions and Implications Zolpidem inhibits cardiac hERG K+ channels. Despite a relatively low affinity of zolpidem to hERG channels, APD prolongation may lead to acquired LQTS and TdP in cases of reduced repolarization reserve or zolpidem overdose. PMID:23061993

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

Experimental research on anchoring force in intestine for the motion of capsule robot.

PubMed

Chen, Wenwen; Ke, Quan; He, Shu; Luo, Weijie; Ji, Xing Chun; Yan, Guozheng

2013-07-01

Multiple research groups are currently attempting to develop less-invasive robotic capsule endoscopes (RCEs) with better outcomes for enteroscopic procedures. Understanding the biomechanical response of the bowel to RCE is crucial for optimizing the design of these devices. For this reason, this study aims to develop an analytical model to predict the anchoring force of the model when travelling through the intestine. Previous work has developed, characterized and tested the frictional characteristics of the intestine with microgroove structures that had different surface contours. This work tested basic anchoring force characteristics with custom-built testers and clamping mechanism dummies to analyse the robot clamping movement (which is vital to improving movement efficiency). Balloon-shaped and leg-based clamping mechanisms were developed, which were found to have variable anchoring forces from 0.01 N to 1.2 N. After analysing the experimental results it was found that: (a) robot weight does not play a major role in anchoring force; (b) an increase in anchoring force corresponded to an increase in diameter of the clamping mechanism; and (c) textured contact surfaces effectively increased friction. These results could be explained by the biomechanical response of the intestine, friction and mucoadhesion characteristics of the small intestine material. With these factors considered, a model was developed for determining anchoring force in the small intestine.

Using computer simulations to determine the limitations of dynamic clamp stimuli applied at the soma in mimicking distributed conductance sources.

PubMed

Lin, Risa J; Jaeger, Dieter

2011-05-01

In previous studies we used the technique of dynamic clamp to study how temporal modulation of inhibitory and excitatory inputs control the frequency and precise timing of spikes in neurons of the deep cerebellar nuclei (DCN). Although this technique is now widely used, it is limited to interpreting conductance inputs as being location independent; i.e., all inputs that are biologically distributed across the dendritic tree are applied to the soma. We used computer simulations of a morphologically realistic model of DCN neurons to compare the effects of purely somatic vs. distributed dendritic inputs in this cell type. We applied the same conductance stimuli used in our published experiments to the model. To simulate variability in neuronal responses to repeated stimuli, we added a somatic white current noise to reproduce subthreshold fluctuations in the membrane potential. We were able to replicate our dynamic clamp results with respect to spike rates and spike precision for different patterns of background synaptic activity. We found only minor differences in the spike pattern generation between focal or distributed input in this cell type even when strong inhibitory or excitatory bursts were applied. However, the location dependence of dynamic clamp stimuli is likely to be different for each cell type examined, and the simulation approach developed in the present study will allow a careful assessment of location dependence in all cell types.

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