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At least 667 records · Page 37Linked to original sources
PubMed1993

Attenuation of hippocampal long-term potentiation by ethanol: a patch-clamp analysis of glutamatergic and GABAergic mechanisms.

Long-term potentiation of synpatic transmission (LTP) of the perforant path--dentate gyrus synapse is induced by 5 Hz, theta-like stimulation patterns. Such stimuli induce plasticity that is most likely driven by a decrease in synaptic inhibition (disinhibition) mediated by GABAB autoreceptors. In the present study, we demonstrate that LTP induced in this manner is completely antagonized by ethanol. In order to determine the site of ethanol inhibition of LTP induced by theta-like stimulation, we combined slice patch recordings with pharmacologic isolation of the individual glutamatergic and GABAergic synaptic currents. The present experiments revealed that ethanol inhibited NMDA receptor-mediated synaptic currents without potentiation of GABAA currents or attenuation of GABAB-mediated fading of GABAA synaptic currents. These observations with ethanol contrasted with the actions of the water-soluble benzodiazepine midazolam, which strongly potentiated GABAA synaptic currents, reversed the effect of GABAB-mediated fading of GABAA synaptic currents, and therefore blocked the resulting NMDA synaptic currents. These data indicate that the effects of ethanol on long-term changes in synaptic strength in the rat hippocampal formation are due primarily to an action at the NMDA receptor-channel complex.

Animals↗
PubMed1988

Development of membrane-based biosensors: measurement of current from photocycling bacteriorhodopsin on patch clamp electrodes.

Our initial work toward developing membrane protein-based biosensors has involved use of bacteriorhodopsin (BR) as a model membrane protein. BR was incorporated into liposomes of a polymerizable lecithin, and was shown to pump protons in response to illumination both before and after polymerization of the lipids. In the work described in this paper, BR was first reincorporated in liposomes of asolectin by consonication with purple membrane. The liposomes, which sustained the function of the protein, were used to form a monolayer at the air-water interface. This monolayer was transferred as a bilayer onto patch electrode. When illuminated with a pulse of 514.5-nm light the lipid/protein patch produced a current spike into the pipette corresponding to events no later than the generation of the 412-nm intermediate, probably caused by pumping of protons across the patch membrane. The experiment demonstrates not only the extreme sensitivity of amperometric detection, but also a small tendency for membrane proteins to preferentially orient in this configuration.

Bacteriorhodopsins↗
PubMed1992

Glass technology for patch clamp electrodes.

Based on all of the properties of glass described here, it is obvious that no one glass can be recommended for all purposes and for all cells. Borosilicate glasses like 7760, 7052, and 7040 are good general purpose glasses for both single-channel and whole-cell recordings. They are good initial choices but, of course, must be checked for each cell type for problems associated with leaching of blockers, etc., from the glass. Corning 8161 is the best glass studied to date with respect to electrical and thermal properties but must be checked carefully for leachable components. If perforated-patch whole-cell recordings are to be used, 8161, KG-12, or some other high lead, low melting point glass are probably the best choices.

Data Collection↗
PubMed1996

Amperometric detection of quantal secretion from patch-clamped rat pancreatic beta-cells.

Serotonin (5-HT) is taken up in insulin granules and co-released with insulin on stimulation of pancreatic islet beta-cells. Based on these observations, we have used microcarbon fiber amperometry to examine secretogogue-induced 5-HT release from rat beta-cells preloaded for 4-16 h with 5-HT and then exposed to a bath solution containing 10 microM forskolin. In response to local application of KCl (60 mM) or tolbutamide (50-200 microM), we recorded barrages of amperometric events. Each amperometric event consisted of a short pulse of current measurable at electrode voltages that catalyze 5-HT oxidation. With either secretogogue, release was calcium-dependent. On combining amperometry with perforated patch whole-cell recording, we found that barrages of such events were well coupled in time and graded in intensity with depolarization-induced Ca2+ currents and well correlated with increases in membrane capacitance. In cell-attached patch recording, amperometric events evoked by application of tolbutamide followed the closure of ATP-sensitive K+ channels and coincided with the onset of electrical activity. These experiments suggest that amperometry is a useful technique for studying, in real time, the dynamic aspects of stimulus-secretion coupling in beta-cells. Their performance was facilitated by the design of a new carbon fiber electrode (ProCFE) described within.

Animals↗
PubMed2005

Permeability of R6G across Cx43 hemichannels through a novel combination of patch clamp and surface enhanced Raman spectroscopy.

We have measured the permeability of rhodamine-6G across Cx43 hemichannels reconstituted on a pipette tip. Cx43 hemichannels were overexpressed in Sf9 cells, and affinity-purified. The hemichannels were reconstituted in a lipid bilayer on a pipette tip by the tip-dip method. R6G in the pipette permeated across the channels into the bath. The permeability of R6G was quantified by measuring R6G concentration in the bath after several hours by surface enhanced Raman spectroscopy (SERS) with 100 nm silver colloid particles. The ratio of the permeability of dye to salt, as extracted by this combined electrical-SERS technique, is compatible with similar ratios for other dyes across whole gap junction channels. The results for the permeability ratio were further compared to fluorescence measurements. The novel combination of patch and SERS techniques can be extended to quantifying the transport of biologically significant non-fluorescent molecules, such as cAMP and IP3, across 1 nm sized pores, such as the gap junction channel.

Journal Article↗
PubMed1986

Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels.

Functional acetylcholine receptor (AChR) and sodium channels were expressed in the membrane of Xenopus laevis oocytes following injection with poly(A)+-mRNA extracted from denervated rat leg muscle. Whole-cell currents, activated by acetylcholine or by depolarizing voltage steps had properties comparable to those observed in rat muscle. Oocytes injected with specific mRNA, transcribed from cDNA templates and coding for the AChR of Torpedo electric organ, expressed functional AChR channels at a much higher density. Single-channel currents were recorded from the oocyte plasma membrane following removal of the follicle cell layer and the vitelline membrane from the oocyte. The follicle cell layer was removed enzymatically with collagenase. The vitelline membrane was removed either mechanically after briefly exposing the oocyte to a hypertonic solution, or by enzyme treatment with pronase. Stretch activated (s.a.) currents were observed in most recordings from cell-attached patches obtained with standard patch pipettes. S.a.-currents were evoked by negative or positive pressure (greater than or equal to 5 mbar) applied to the inside of the pipette, and were observed in both normal and mRNA injected oocytes indicating that they are endogenous to the oocyte membrane. The s.a.-channels are cation selective and their conductance is 28 pS in normal frog Ringer's solution (20 +/- 1 degree C). Their gating is voltage dependent, and their open probability increases toward more positive membrane potentials. The density of s.a.-channels is estimated to be 0.5-2 channels per micron 2 of oocyte plasma membrane. In cell-attached patches s.a.-currents are observed much less frequently when current measurement is restricted to smaller patches of 3-5 micron 2 area using thick-walled pipettes with narrow tips. In outside-out patches s.a.-currents occur much less frequently than in cell-attached or inside-out patches. AChR-channel and sodium channel currents were observed only in a minority of patches from oocytes injected with poly(A)+-mRNA from rat muscle. AChR-channel currents were seen in all patches of oocytes injected with specific mRNA coding for Torpedo AChR. In normal frog Ringer's solution (20 +/- 2 degrees C) the conductance of implanted rat muscle AChR-channels was 38 pS and that of sodium channels 20 pS. The conductance of implanted Torpedo AChR channels was 40 pS. The conductance of implanted channels was similar in cell-attached and in cell-free patches.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

The light-sensitive conductance of hyperpolarizing invertebrate photoreceptors: a patch-clamp study.

Tight-seal recording was employed to investigate membrane currents in hyperpolarizing ciliary photoreceptors enzymatically isolated from the eyes of the file clam (Lima scabra) and the bay scallop (Pecten irradians). These two organisms are unusual in that their double retinas also possess a layer of depolarizing rhabdomeric cells. Ciliary photoreceptors from Lima have a rounded soma, 15-20 microns diam, and display a prominent bundle of fine processes up to 30 microns long. The cell body of scallop cells is similar in size, but the ciliary appendages are modified, forming small spherical structures that protrude from the cell. In both species light stimulation at a voltage near the resting potential gives rise to a graded outward current several hundred pA in amplitude, accompanied by an increase in membrane conductance. The reversal potential of the photocurrent is approximately -80 mV, and shifts in the positive direction by approximately 39 mV when the concentration of extracellular K is increased from 10 to 50 mM, consistent with the notion that light activates K-selective channels. The light-activated conductance increases with depolarization in the physiological range of membrane voltages (-30 to -70 mV). Such outward rectification is greatly reduced after removal of divalent cations from the superfusate. In Pecten, cell-attached recordings were also obtained; in some patches outwardly directed single-channel currents could be activated by light but not by voltage. The unitary conductance of these channels was approximately 26 pS. Solitary ciliary cells also gave evidence of the post stimulus rebound, which is presumably responsible for initiating the "off" discharge of action potentials at the termination of a light stimulus: in patches containing only voltage-dependent channels, light stimulation suppressed depolarization-induced activity, and was followed by a strong burst of openings, directly related to the intensity of the preceding photostimulation.

Action Potentials↗
PubMed1991

Patch clamp analysis of excitatory synaptic currents in granule cells of rat hippocampus.

1. Excitatory postsynaptic potentials (EPSPs) and their underlying currents (EPSCs) were recorded from dentate granule cells in thin hippocampal slices of rats using the tight-seal whole-cell recording technique. 2. At resting membrane potentials (ca -60 to -70 mV), the EPSCs clearly consisted of a dominant fast and a smaller slow component. The slow EPSC component markedly increased with depolarization. This resulted in a region of negative slope conductance (between -50 and -30 mV) in the peak current-voltage (I-V) relation of the dual-component EPSC in most neurones. The EPSCs reversed entirely at -1.2 +/- 2.8 mV (n = 15). 3. Using selective antagonists of N-methyl-D-aspartate (NMDA) and non-NMDA excitatory amino acid receptors, two pharmacologically distinct components of the natural EPSCs were isolated. The non-NMDA EPSCs displayed a linear I-V relation. Their rise times (0.5-1.9 ms) were independent of membrane voltage but seemed to depend critically on the precise dendritic location of the synapse. Their decay was approximated by a single exponential with a time constant ranging from 3 to 9 ms. The time course of these EPSCs was independent of changes in extracellular Mg2+. 4. The NMDA EPSCs displayed a non-linear I-V relation. At resting membrane potentials their peak amplitudes were 20 pA and increased steadily with depolarization to -30 mV. At membrane voltages positive to -30 mV the peak I-V relation was linear. The rise times of NMDA EPSCs ranged from 4 to 9 ms and were insensitive to membrane voltage. 5. The NMDA EPSCs decayed biexponentially. Both time constants, tau f and tau s, increased with depolarization in an exponential manner, tau s being more voltage dependent than tau f. Lowering extracellular Mg2+ slightly reduced both rate constants but did not completely abolish their voltage sensitivity. 6. Bath application of NMDA to outside-out patches from granule cells induced single channel currents of 52 pS in nominally Mg(2+)-free solutions. They displayed a burst-like single-channel activity with clusters of bursts lasting several hundreds of milliseconds. Currents through single NMDA receptor channels reversed around 0 mV. 7. The fractional contributions of NMDA and non-NMDA components to peak currents and synaptic charge transfer were assessed. At resting membrane potential the NMDA EPSC component accounted for 23% of the peak current and for 64% of the synaptic charge transfer. The contribution of the NMDA EPSC component to the synaptic charge transfer strongly increased with small depolarizations from rest.

Animals↗
PubMedDate not supplied

Use of the cell-attached patch clamp technique to examine regulation of single cardiac K channels by cyclic GMP.

Cyclic nucleotides play a central role in the modulation of ion channels in a variety of tissues, including the heart. In order to determine the possible role of cyclic GMP (cGMP) in the regulation of the background K channel activity of cardiac cells, the effect of 8-Br-cGMP on the inwardly-rectifying K channels of cultured ventricular myocytes from embryonic chick hearts was examined. 8-Br-cGMP (10(-4) to 10(-3) M) inhibited these single channel currents within 3 to 10 min. Spontaneous recovery of the currents occurred with prolonged (greater than or equal to 15 min) exposure to 8-Br-cGMP, but this recovery was accompanied by altered channel behavior. Thus, a new long-lasting open state of the channel appeared, in addition to the open state observed prior to 8-Br-cGMP addition. Superfusion of the cells with the muscarinic agonist carbamylcholine (10(-5) M) also resulted in inhibition of the currents, which suggests that the cGMP-mediated inhibition of these channels may occur under physiological conditions. Thus, it appears that cGMP may be an important modulator of the background K conductance (and excitability) of cardiac cells.

Animals↗
PubMed1992

A patch-clamp study of the Ca2+ mobilization from internal stores in bovine aortic endothelial cells. II. Effects of thapsigargin on the cellular Ca2+ homeostasis.

Evidence was provided, in the preceding paper (Thuringer & Sauvé, 1992), that the external Ca(2+)-dependent phase of the Ca2+ signals evoked by bradykinin (BK) or caffeine in bovine aortic endothelial cells (BAE), differ in their respective sensitivity to procaine. To examine whether the emptying of the InsP3-sensitive Ca2+ store is the signal for activating the agonist-evoked Ca2+ entry, we have investigated the effects of thapsigargin (TSG), a known inhibitor of the microsomal Ca(2+)-ATPase activity in a variety of cell types, via the activity of calcium-activated potassium channels [K(Ca2+) channels]. In cell-attached experiments, the external application of TSG caused a sustained or oscillatory activation of K(Ca2+) channels depending on both the cells and doses tested. The TSG-evoked channel activity could be reversibly blocked by removing extracellular Ca2+, and strongly decreased by adding 10 mM procaine to the bath medium. In Ca(2+)-free external conditions, TSG did not promote an apparent Ca2+ discharge from internal stores but prevented in a dose- and time-dependent manner the subsequent agonist-evoked channel activity related to the release of internally sequestered Ca2+. These results confirm that TSG and BK release Ca2+ from the same internal stores but with different kinetics. Because the channel response to caffeine was found to be poorly sensitive to procaine, in contrast to that evoked by BK and TSG, it may be concluded that both BK and TSG activate the same Ca2+ entry pathway. Therefore, the emptying of the InsP3-sensitive Ca2+ store is likely to be the main signal for activating the agonist-evoked Ca2+ entry in BAE cells.

Animals↗
PubMed1993

A nonlinear filter algorithm for the detection of jumps in patch-clamp data.

This paper presents a new algorithm for the detection of channel openings and closures from noisy current signals, especially for the correct generation and interpretation of open-time and closed-time histograms. The Hinkley detector is a nonlinear off-line jump detection algorithm from the field of fault detection. Here, an improved version, the higher-order Hinkley detector H.O.H.D., is developed. A general description of the sensitivity of a detector is introduced by the time resolution tres. This allows a comparison of the nonlinear detectors with the standard threshold detectors preceded by a low-pass filter for noise suppression. By means of application to simulated and real data, the performance of the detection algorithms is investigated. The higher-order Hinkley detector gave the best results with respect to correct reconstruction of the event length, to a small amount of missed brief events as well as to the ability to achieve short time resolution without pretending false events, especially in the presence of colored noise.

Algorithms↗
PubMed1990

A direct-reading device for measurement of patch-clamp micropipette tip diameters.

This paper describes an electronic device for the measurement of the diameter of the tip opening of micropipettes. The device measures the threshold pressure required to produce bubbles from the micropipette submerged in a liquid and displays the tip inner diameter in micrometres. We demonstrate that this technique can be used effectively in a range of liquids of different density, surface tension and viscosity and that the thickness of the glass from which the micropipettes are made does not influence the measurements.

Electronics↗
PubMed1988

Patch-clamp techniques for time-resolved capacitance measurements in single cells.

Two methods are described for estimation of passive cell parameters such as membrane capacitance, membrane conductance and access resistance in tight-seal whole cell recording. Both methods are restricted in their application to cases where the cell under study can be approximated by a simple three-component network with linear properties over some voltage range. One method, referred to as the time domain technique, requires only standard electrophysiological equipment and a computer. Parameters are derived from an analysis of capacitive transients during square wave stimulation. It is readily adaptable to wide variations in experimental parameters. Particularly, it is equally applicable to the "slow whole-cell" configuration (access resistance in the range 100 M omega to 1 G omega) and to normal whole-cell measurements (access resistance typically 10 M omega). The other method applies a sine wave command signal to the cell and employs a lock-in amplifier to analyse the resulting current signal. Two modes of operating the lock-in amplifier are described. One mode provides an output signal directly proportional to small changes in capacitance at maximum resolution (1-10 fF). The other mode, in conjunction with a digital computer, supplies estimates of all passive cell parameters, as does the time domain technique, but with a large amount of data reduction performed by the lock-in amplifier itself. Due to the special hardware, however, this method is not as flexible as the time domain technique.

Animals↗