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At least 127 records · Page 7Linked to original sources

PubMed1995

Patch-clamping in brain slices: synaptic transmission from ATP to long-term potentiation.

Application of patch-clamp techniques to brain slices has resulted in an enormous increase in the resolution of synaptic currents in mammalian central neurones. This improved resolution has allowed direct observation of miniature and evoked synaptic currents, leading, in agreement with other findings, to the conclusion that the quantal size of synaptic currents in the brain is limited by the number of postsynaptic receptors. Possible explanations for the skewed miniature distribution, observed at all fast central synapses are discussed, with reference to anatomical observations. As a result, a model is proposed which is consistent with much of the apparently contradictory data on the induction and maintenance of long-term potentiation. In addition to the study of mechanisms of synaptic transmission, improved resolution provided by the patch-clamp technique has allowed resolution of synaptic currents, in the brain, mediated by ATP. The role of ATP as a central neurotransmitter is discussed in terms of the above findings.

Adenosine Triphosphate↗

PubMed1998

Patch-clamp detection of macromolecular translocation along nuclear pores.

The present paper reviews the application of patch-clamp principles to the detection and measurement of macromolecular translocation along the nuclear pores. We demonstrate that the tight-seal 'gigaseal' between the pipette tip and the nuclear membrane is possible in the presence of fully operational nuclear pores. We show that the ability to form a gigaseal in nucleus-attached configurations does not mean that only the activity of channels from the outer membrane of the nuclear envelope can be detected. Instead, we show that, in the presence of fully operational nuclear pores, it is likely that the large-conductance ion channel activity recorded derives from the nuclear pores. We conclude the technical section with the suggestion that the best way to demonstrate that the nuclear pores are responsible for ion channel activity is by showing with fluorescence microscopy the nuclear translocation of ions and small molecules and the exclusion of the same from the cisterna enclosed by the two membranes of the envelope. Since transcription factors and mRNAs, two major groups of nuclear macromolecules, use nuclear pores to enter and exit the nucleus and play essential roles in the control of gene activity and expression, this review should be useful to cell and molecular biologists interested in understanding how patch-clamp can be used to quantitate the translocation of such macromolecules into and out of the nucleus.

Biological Transport↗

PubMed2003

Stabilization of high-resistance seals in patch-clamp recordings by laminar flow.

The formation of a high-resistance electrical seal between a cell membrane and a glass micropipet tip is essential in patch-clamp experiments. We have studied the electrical properties and the mechanical stability of the seal using a microfluidic chip generating laminar flow in open volumes. We show that, by using fluid flow (1-10 mm/s) acting along the symmetry axis of the cell-pipet, seals of a higher mechanical stability with increased resistances can be achieved, allowing up to 100% longer recording times and over 40% decreased noise levels (Irms). These improved properties are beneficial for high-sensitivity patch-clamp recordings, in particular, in longtime studies of ion channel receptor systems that are relevant in biosensor applications of the technique. Furthermore, these observations support the combination of patch-clamp with microfluidic devices, for example, for rapid solution exchange around a single cell sensor for high-throughput electrophysiology and for highly resolved kinetic studies.

Journal Article↗

PubMed2004

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

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.

Algorithms↗

PubMed1996

Patch-clamp study on membrane properties and transmitter activated currents of rabbit area postrema neurons.

Using the patch-clamp technique in combination wit sliced tissue preparation the membrane properties of newborn rabbit area postrema neurons were investigated. The neurons responded upon depolarization with a fast Na+-current followed by an inactivating and non-inactivating K+-current. GABA-activated currents were investigated resulting in a large C1-(-)conductance, indicating the expression of GABAA-receptors. The expression of glutamate receptor mRNA was studied by in situ hybridization and electrophysiological measurements of these receptors by means of the patch-clamp technique. As a main result it was found that ionotropic glutamate receptors in the area postrema are composed of "flop" variants of the GluA-, GluB- and GluC-subunits.

Animals↗

PubMed1986

Patch clamp technique and biophysical study of membrane channels.

The present work describes the patch clamp technique, which first allowed the recording of single channel currents in biological membranes. In particular, it describes procedures for preparation and applications of the four different patch clamp configurations. Briefly, the cell-attached configuration is widely used for investigating channel modulation by transmitters acting via second messengers. The cell-free configurations (inside-out and outside-out), complementary to one another with respect to the orientation of the membrane surface, are particularly indicated for the study of the biophysics (kinetics, conductivity, selectivity, mechanism of permeation and block) of ionic channels. Finally, the whole-cell configuration which, because of the remarkable feature that it allows voltage clamp of very small cells, has given access to a number of physiologically important preparations never studied before.

Animals↗

PubMed2004

Visually guided patch-clamp recording of spinal dorsal horn neuron's postsynaptic current evoked by primary afferent fiber.

The authors describe here the procedures for using the gelatin half-embedding method to obtain thin spinal cord slices with attached dorsal roots and performing visually guided whole-cell patch-clamp recording of postsynaptic currents evoked by primary afferent fibers in rat spinal dorsal horn. A segment of spinal cord with attached dorsal roots was prepared and half-embedded in an agar block with 20% (w/v) gelatin. Thin spinal cord slices with attached dorsal roots were obtained with a vibratome and whole-cell patch-clamp configuration was established under the infrared observation. At the holding potential of -70 mV, spontaneous excitatory postsynaptic currents (EPSCs) and dorsal root stimulation-evoked EPSCs were recorded as inward currents. According to the conduction velocity of afferent fibers and stimulus threshold, evoked EPSCs that are mediated by A-like or C-like fibers were distinguished. At the holding potential of 0 mV, spontaneous inhibitory postsynaptic currents (IPSCs) and dorsal root stimulation-evoked IPSCs were recorded as outward currents. Using 5 micromol/L strychnine or 20 micromol/L bicuculline, GABAergic or glycinergic evoked IPSCs could be isolated. Using visual patch-clamp method synaptic transmission can be accurately assessed by measuring postsynaptic currents of the dorsal horn neurons. More importantly, with the aid of infrared observation, the incidence of failure to establish a clamp configuration can be greatly reduced and it becomes easier to make recordings from the neurons in deep dorsal horn laminae. Thus, the present research approach an effective approach to study the modulation of primary afferent synaptic transmission.

Animals↗

PubMed2003

Scientific and technological aspects of oxygen-sensitive electrodes for measurements of oxygen partial pressure in patch-clamp experiments.

Anoxia/hypoxia induces dramatic changes in brain activity leading to the damages in brain structure. Several minutes of decrease in environmental oxygen partial pressure (P(O2)) can irreversibly destroy nerve cells. Therefore, investigations of intracellular mechanisms responsible for hypoxia-induced changes of mammalian nerve system are very important. On-line adjustment and measurements of P(O2) in bath solution during patch-clamp experiments are especially topical. At the recent time, a special interest on the on-line measurements of oxygen contents in bath solution has appeared as a result of application of old approaches previously used for polarographic measurements of oxygen contents in the blood and tissues. Here we describe the simple method of manufacturing oxygen-sensitive microelectrodes, which can be used with standard patch-clamp amplifier. We also describe the main principles of polarographic method and properties of oxygen-sensitive electrodes used in patch-clamp experiments.

Biophysics↗

PubMed2003

Patch clamp analysis of a H+ pump heterologously expressed in giant yeast vacuoles.

Despite the usefulness of the patch-clamp technique, its application to ion pumps and transporters in biomembranes is limited. We developed a novel method for determining the activity of a proton-pumping pyrophosphatase (H(+)-PPase) made of a single protein. We heterologously highly expressed the enzyme in Saccharomyces cerevisiae, prepared giant vacuoles from the cells, and measured a PPi-dependent electrical current of 18 pA (10.5 fA/ micro m(2)) using the patch-clamp technique in the whole-vacuole recording mode. We determined the inhibitor sensitivity and affinity for substrate (K(m), 4.6 micro M). The enzyme number in a giant vacuole (4.2 x 10(6)) and the molecular activity of the expressed H(+)-PPase (14 s(-1)) were determined. An uncoupling-type H(+)-PPase mutant, of which the 263rd glutamate residue was replaced by aspartate, and of which H(+) pump activity was not detected with the fluorescence quenching method, showed a weak current with a high K(m). The high accuracy, effectiveness and applicability of the method for exogenously expressed ion transporters were also discussed.

Biochemical Phenomena↗

PubMed2003

Probing conformational changes of gramicidin ion channels by single-molecule patch-clamp fluorescence microscopy.

Complex conformational changes influence and regulate the dynamics of ion channels. Such conformational changes are stochastic and often inhomogeneous, which makes it extremely difficult, if not impossible, to characterize them by ensemble-averaged experiments or by single-channel recordings of the electric current that report the open-closed events but do not specifically probe the associated conformational changes. Here, we report our studies on ion channel conformational changes using a new approach, patch-clamp fluorescence microscopy, which simultaneously combines single-molecule fluorescence spectroscopy and single-channel current recordings to probe the open-closed transitions and the conformational dynamics of individual ion channels. We demonstrate patch-clamp fluorescence microscopy by measuring gramicidin ion channel conformational changes in a lipid bilayer formed at a patch-clamp micropipette tip under a buffer solution. By measuring single-pair fluorescence resonance energy transfer and fluorescence self-quenching from dye-labeled gramicidin channels, we observed that the efficiency of single-pair fluorescence resonance energy transfer and self-quenching is widely distributed, which reflects a broad distribution of conformations. Our results strongly suggest a hitherto undetectable correlation between the multiple conformational states of the gramicidin channel and its closed and open states in a lipid bilayer.

Anti-Bacterial Agents↗

PubMed1988

Patch-clamp recording of amino acid-activated responses in "organotypic" slice cultures.

Patch-clamp recording techniques were used to study the properties of amino acid-activated channels in cultured "organotypic" slices from rat cerebellum and hippocampus. Hippocampal pyramidal cells responded to the three main glutamatergic agonists, N-methyl-D-aspartate (N-Me-D-Asp), quisqualate, and kainate, whereas Purkinje cells responded only to quisqualate and kainate. Analysis of single-channel events recorded in outside-out patches from hippocampal neurons showed large conductance events (50 pS), which occurred more frequently in the presence of glycine. These events could be produced by N-Me-D-Asp and also, at low frequency, by quisqualate. On the other hand, 50-pS events were never observed in Purkinje neurons. This supports the hypothesis that N-Me-D-Asp and "non-N-Me-D-Asp" receptors are distinct molecular entities. Comparison of whole-cell and outside-out patch recordings from Purkinje cells revealed a clear spatial segregation of gamma-aminobutyric acid (GABA) and glutamate receptors: although GABA receptors are found at high density in somatic membrane, quisqualate and kainate receptors are mostly extrasomatic. The results show that organotypic slice cultures are amenable to patch-clamp methods. They also show that, in these cultures, amino acids receptors have specific distribution patterns according to cell type and to region within a cell.

Action Potentials↗

PubMed2002

What can we learn about cell signalling by combining optical imaging and patch clamp techniques?

Optical imaging is a powerful technique with which to investigate the activity, distribution and movement of biomolecules. The increased resolution of images obtained with confocal microscopy now allows us to visualize the signalling events in individual intracellular organelles. Local photobleaching and uncaging of caged compounds enable investigators to control the activity of many biologically important molecules in small localized regions of both cytosol and internal spaces of cellular organelles. Uncaging and photobleaching conveniently complement laser scanning confocal microscopy. The whole-cell recording configuration of the patch-clamp technique has been widely used not only to measure ionic currents, but also to control the concentration of important molecules in the cytosol. The cell-attached configuration of patch clamp was utilized for local stimulation of the cell and local delivery of the second messengers. This paper describes the advantages of combining patch-clamp and optical imaging methods as well as some of the recent achievements using this approach.

Calcium Signaling↗

PubMed2001

A patch-clamp study on human sperm Cl- channel reassembled into giant liposome.

AIM: To record the single-channel currents and characterize the electrophysiological properties of the Cl- channels in human sperm membrane. METHODS: The membrane proteins extracted from the human sperm were reassembled into liposome bilayer, and the liposomes were fused into giant liposomes with a diameter more than 10 microm by dehydration-rehydration procedure. The giant liposomes were used to study the Cl- channel activities by patch-clamp technique. RESULTS: By patch clamping the giant liposome in an asymmetric NMDG (N-methyl-D-glucamine)-Cl (bath 100// pipette 200 mmol/L) solution system, three kinds of single-channel events with unit conductances of (74.1 +/- 8.3) pS, (117.0 +/- 5.7) pS and (144.7 +/- 4.5) pS, respectively, were detected. Their activities were voltage-dependent and all were blocked by SITS (4-acetamido-4'-isothiocyanato-stilbene-2', 2'-disulfonic acid) in a concentration-dependent manner. By constructing the open and close dwell time distribution histograms and then fitting them with exponential function, two time constants were obtained in both the open and the close states. The burst activity and conductance substate of the channels were observed. CONCLUSION: There exist three kinds of Cl- channels with different conductance in human sperm membrane at least.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMedDate not supplied

A whole-cell patch clamp technique which minimizes cell dialysis.

A variant of the whole-cell patch clamp technique is described which allows measurement of whole-cell ionic currents in small cells while minimizing cell dialysis with the pipette solution. The technique involves the application of negative pressure to the inside of small (less than 1 micron) tip diameter pipettes placed on the cell surface to achieve high resistance seals and membrane rupture. The technique has been used successfully in a variety of different types of cells to study membrane currents carried by Ca and K, currents generated by exchange carriers as well as electrical coupling between cells. Overall, the technique seems well suited for the study of ionic currents in small cells, and provides an alternative to conventional patch clamping techniques which necessitate intracellular dialysis.

Action Potentials↗

PubMed2005

Improved superfusion technique for rapid cooling or heating of cultured cells under patch-clamp conditions.

We have developed an improved technique for fast cooling and heating of solutions superfusing isolated cells under patch-clamp or calcium imaging conditions. The system meets the requirements for studying temperature dependency of all kinds of ion channels, in particular temperature-gated ion channels. It allows the application of temperature changes within a range of 5-60 degrees C at maximum rates of -40 degrees C/s to 60 degrees C/s. Barrels filled with different solutions are connected to a manifold consisting of seven silica capillaries (320 microm inner diameter, i.d.). A common outlet consists of a glass capillary through which the solutions are applied onto the cell surface. The upper part of this capillary is embedded in a temperature exchanger driven by a miniature Peltier device which preconditions the temperature of the passing solution. The lower part of the capillary carries an insulated copper wire, densely coiled over a length of 7 mm, and connected to a dc current source for resistive heating. The Peltier device and the heating element are electrically connected to the headstage probe which is fixed on to a micromanipulator for positioning of the manifold. The temperature of the flowing solution is measured by a miniature thermocouple inserted into the common outlet capillary near to its orifice which is placed at a distance of less than 100 microm from the surface of the examined cell. The temperature is either manually controlled by voltage commands or adjusted via the digital-to-analog converter of a conventional data acquisition interface. Examples are given of using the device in patch-clamp studies on heterologously expressed TRPV1, TRPM8, and on cultured rat sensory neurons.

Action Potentials↗

PubMed1995

PCR and patch-clamp analysis of single neurons.

The combination of patch-clamp and molecular biology techniques has made it possible to characterize the pharmacological and biophysical properties of ion channels in single neurons and to screen for expression of specific mRNAs in the same cell. Following whole-cell recording, the cytoplasm of the cell is harvested, and RNA is reverse transcribed into cDNA and amplified in PCR with primers specific for individual ion channel subunits. Additional experiments can then be designed to relate structure and function at the protein level more directly, since cells appear to regulate the composition of ion channels at least partly at the posttranscriptional stage.

DNA Primers↗

PubMed2004

Analysis of receptive fields revealed by in vivo patch-clamp recordings from dorsal horn neurons and in situ intracellular recordings from dorsal root ganglion neurons.

It has been thought that spinal dorsal horn neurons receive convergent inputs from not only somatosensory but also visceral pathways. For instance, the referred pain is presumed to be due to the convergence of sensory inputs from cardiac and shoulder receptive fields. However, precise investigation has not been made from dorsal horn neurons yet, because of difficulty in studying the pathways from those regions by means of conventional electrophysiology. The purpose of this study is to clarify the convergent inputs to single dorsal horn neurons from wide receptive fields using an in vivo patch-clamp recording technique from the superficial spinal dorsal horn and an intracellular recording from dorsal root ganglion neurons that keep physiological connections with the peripheral sites. Identified dorsal root ganglion neurons received an input from a quite small area, about 1 x 1 mm in width of the skin. In contrast, substantia gelatinosa neurons in the spinal cord received inputs from an unexpectedly wide area of the skin. Previous extracellular recordings have, however, revealed that substantia gelatinosa neurons have small receptive field. This discrepancy is probably due mainly to an availability of the in vivo patch-clamp method to analyze sub-threshold synaptic responses. In contrast, the extracellular recording technique allows us to analyze predominantly the firing frequency of neurons. Thus, the in vivo patch-clamp recordings from dorsal horn neurons and the intracellular recordings from DRG neurons will be useful for well understanding the sensory processing in the spinal cord.

Animals↗

PubMed2006

Detection of SEB gene by bilayer lipid membranes nucleic acid biosensor supported by modified patch-clamp pipette electrode.

This work reports a kind of novel bilayer lipid membranes (BLMs) nucleic acid biosensor supported by modified patch-clamp pipette electrode was developed to detect staphylococcus enterotoxins B (SEB) gene. BLMs were formed within 15 min and able to be operated at least 24 h. Hydrophobic dodecane tail (C12) modified 18 bp single-stranded DNA (ssDNA) probe was immobilized on BLMs. The electrochemical currents versus the different concentration of ssDNA probe immobilized on BLMs indicated linearly correlation. The BLMs nucleic acid biosensor was fabricated by selecting the ssDNA probe as the signal sensing element with the concentration of 273.65 ng/mL. The electrochemical performance of the biosensor for the detection of SEB was investigated. The result showed that linear relationship was found between the current and ln(concentration) from 20 to 5000 ng/mL and the detection limit was 20 ng/mL. In addition, the biosensor was specific response to SEB gene and showed no significant current alteration in electrolyte which containing no SEB gene. Finally, Atom Force Microscope (AFM) images could be observed and used to evaluate the superficial microstructure of BLMs, ssDNA immobilized on BLMs and BLMs after hybridization. The BLMs nucleic acid biosensor supported by modified patch-clamp pipette electrode will become a highly sensitive, rapid, selective analytical tool for detection of Staphylococcus aureus, which produce SEB.

Biosensing Techniques↗