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At least 1,495 records · Page 83Linked to original sources
PubMed1992

A loudspeaker-driven system for rapid and multiple solution exchanges in patch-clamp experiments.

A new and inexpensive system allowing rapid and synchronized changes of solutions around a membrane patch or a cell under voltage-clamp conditions is described. Four plastic capillary tubings (OD 640 microns; ID 430 microns) were glued together horizontally and attached to a coil of a commercially available loudspeaker. Servo-control of the position of the coil allowed the mouth of any of the capillaries to be positioned near the pipette tip within 6 ms. A high flow speed of the test solution was crucial to achieve rapid solution exchange. At a flow speed of 5 cm/s, complete exchange of the external environment of a frog ventricular cell was achieved within 20-30 ms. The time course of solution change was found to be 3-5 times faster at the tip of an open patch pipette. To preserve the physical integrity of the cell, the cell was usually perfused by a control capillary at a slow velocity (0.2-0.4 cm/s) and test solutions flowing out of adjacent capillaries at high velocity (4-5 cm/s) were applied to the cell only for short periods. Determination of the three-dimensional contamination profile around the mouth of the control capillary allowed the optimal conditions for the use of the system to be established and possible sources of contamination to be avoided between adjacent capillaries with unmatched flow speeds. Successive and multiple changes in external solutions could be easily synchronized with voltage-clamp depolarizations to examine the time course of the effect of drugs on voltage-operated ion channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy.

A description is given of the implementation of infrared differential interference contrast (IR-DIC) video microscopy to an upright compound microscope. Using the improved resolution offered by IR-DIC a procedure is described for making patch-pipette recordings from visually identified neuronal somata and dendrites in brain slices. As an example of the application of this technique to electrophysiological recordings from small neuronal processes in brain slices we describe whole-cell current-clamp and cell-attached and excised patch-clamp recordings from the apical dendrites of layer V pyramidal neurons in slices of rat neocortex.

Animals↗
PubMed1993

Effect of various diuretics on membrane voltage of macula densa cells. Whole-cell patch-clamp experiments.

The tubuloglomerular feedback mechanism is inhibited by diuretics such as furosemide. For the macula densa (MD) cells similar transport systems, as present in thick ascending limb (TAL) cells, have been suggested. To examine this further, membrane voltages (Vm) of MD cells were recorded with the fast or slow whole-cell patch-clamp method. The effects of diuretics on voltages and the conductance properties of these cells were examined. Vm of MD cells measured with the whole-cell patch-clamp method were as high as those in TAL cells: -72 +/- 1 mV (n = 21). An increase in the extracellular K+ concentration by 15 mmol/l depolarized Vm of MD cells by 11 +/- 1 mV (n = 18). Ba2+ (1 mmol/l) reversibly depolarized MD cells by 10 +/- 2 mV (n = 10). Thus, MD cells possess a K+ conductance that could allow for the recycling of K+ taken up by the Na(+)-2 Cl(-)-K+ cotransporter. MD cells hyperpolarized reversibly upon addition of the loop diuretics furosemide, piretanide and torasemide, whereas muzolimine and hydrochlorothiazide, neither one acting on this cotransport system in other preparations including the TAL, had no effect on Vm. MD cells most likely possess the same cotransport system as the TAL cells, which drives NaCl reabsorption in the TAL and serves as sensor for the tubular NaCl concentration in MD cells.

Animals↗
PubMed1982

Posterior atlanto-axial fusion: a new compression clamp for laminar osteosynthesis.

A new method of dorsal arthrodesis in patients with atlanto-axial instability of different etiology is described. In three patients solid laminar osteosynthesis C1/C2 was achieved by insertion of an autogenous cortico-cancellous bone graft into the interlaminar space and by a bilateral application of the lately developed metal clamp. The technical principles of the clamp guarantee a permanent pressure on the graft and a solid initial stability in the segment. Additional external fixation and immobilization of the patient are not necessary. Complications as known from other procedures of the atlanto-axial dorsal fusion will be prevented efficiently.

Adolescent↗
PubMed1993

Beta-adrenergic stimulation does not regulate Na pump function in voltage-clamped ventricular myocytes of the rat heart.

Na pump current was measured in rat ventricular myocytes to determine if beta-adrenergic stimulation can directly modulate Na,K-ATPase activity. Enzymatically-isolated heart cells were voltage-clamped with a single patch electrode and Na pump current was briefly activated by rapidly increasing extracellular [K+] from 0 to 15 mM for 3-5 s after other ionic currents were blocked or inactivated. The salt solution in the voltage-clamping electrode included (in mM): (1) 100 Na+, 10 EGTA, (2) 5 Na+, 10 EGTA, or (3) 100 Na+, 7.5 Ca2+, 10 EGTA (free [Ca2+] = 480 nM). With all three electrode solutions, Na pump current was not significantly changed after 2-4 min in the presence of 10 microM isoprenaline. beta-adrenergic pathways were still intact as evidenced by the two-fold increase in Cd(2+)-sensitive Ba2+ current through calcium channels that was observed in the presence of isoprenaline. Thus, beta-adrenergic stimulation does not appear to directly regulate Na,K-ATPase activity in rat ventricular myocytes.

Adrenergic beta-Agonists↗
PubMed1990

Ionic currents contributing to the action potential in single ventricular myocytes of the guinea pig studied with action potential clamp.

With the action potential clamp procedure we studied the contribution of various ionic currents to the action potential in single ventricular myocytes. Action potentials were elicited by a current pulse through the suction pipette and recorded by a computer. A representative action potential was then repetitively replayed to the same cell under voltage-clamp conditions. Successive pharmacological blocks of ionic currents allowed for the first time the measurement of the contribution of the L-type calcium current (ICa) and the [Ca2+]i-activated currents as well as the potassium current to the action potential. Experiments using caffeine as a tool to increase calcium release from the sarcoplasmic reticulum supported the idea that INaCa contributes to the plateau during the second half of the action potential and even lasts into diastole, whereas strong elevation of the intracellular [Ca]i during the action potential additionally activated the non-specific cation channel.

Action Potentials↗
PubMed1989

Patch clamp experiments on nicotinic acetylcholine receptor-ion channels in bullfrog sympathetic ganglion cells.

Nicotinic acetylcholine-receptor ion channels (AChR channels) were studied in bullfrog sympathetic ganglion cells cultured for 1 day to 3 weeks, using a patch clamp technique. Microsuperfusion of ACh (2-10 microM) to the ganglion cell under the whole cell clamp produced an inward current at membrane potentials negative to -60 mV, which had a fast onset and decay. This rapid ACh-induced current was accompanied by a large current fluctuation, decreased and increased in amplitude by membrane depolarization and hyperpolarization, respectively, and blocked by d-tubocurarine. Thus, this current must be induced by the nicotinic action of ACh, but not by a muscarinic effect to activate a slow cation-selective current. At depolarized levels more than -50 mV, ACh induced an additional inward current which was slow in time course, accompanied by no or decreased current fluctuation and increased in amplitude by membrane depolarization. Accordingly, this slow ACh-induced current could result from the suppression of a voltage-dependent K+ current (M-current: Brown and Adams 1980) by the muscarinic action of ACh. Fluctuation analysis of the rapid ACh-induced current at potentials negative to -50 mV revealed the elementary conductance of 14 pS and a power spectral density distribution of the double Lorentzian function which yielded the time constants of 5.4 and 62.5 ms at -60 to -80 mV. The variance of either component was independent of the mean current.

Acetylcholine↗
PubMed1987

Characterization of calcium-activated potassium channels in single smooth muscle cells using the patch-clamp technique.

Single-channel currents were recorded with the patch-clamp technique from freshly dissociated vertebrate smooth muscle cells from the stomach of Bufo marinus. Of the variety of channels observed, one displayed a large linear conductance of 250 pS (in symmetric 130 mM KCl) which in excised patches was shown to be highly K+ selective. The probability of the channel being open (Po) increased when [Ca2+]i was elevated and/or when the membrane potential was made more positive. Thus, the features of this channel resemble the large-conductance Ca2+-activated K+ channel found in a wide variety of cell types. The voltage sensitivity of the channel was studied in detail. For patches containing a single large-conductance channel a plot of Po versus membrane potential followed the Boltzmann relationship. Increasing [Ca2+]i shifted this plot to the left along the voltage axis to more negative potentials. Both the mean closed time and mean open time varied with potential as a single exponential with almost all of the voltage sensitivity of Po residing in the mean closed time. These results were verified with a series of experiments carried out at low Po (less than 0.1) in patches containing multiple (N) large-conductance channels. Here the ln (NPo) was a linear function of potential with an inverse slope of 9 mV. Almost all of the potential sensitivity lay in the mean closed time the natural log of which was also a linear function of potential with an inverse slope 11 mV in magnitude. The characteristics of this channel as well as the appearance of several of them in almost every patch suggest that they underlie the large peak outward macroscopic current found with whole-cell voltage-clamp studies.

Animals↗
PubMed1987

A new oil-gap method for internal perfusion and voltage clamp of single cardiac cells.

(1.) We designed a new technique to achieve fast voltage clamp, combined with internal perfusion. The single guinea-pig cardiac cell, dissociated by collagenase treatment, was stretched across an oil-gap (30-40 micron wide) from a pool of Tyrode solution to a pool of internal solution. Part of the cell membrane was disrupted in the internal solution by crushing on the cell, a tapered tip of a glass capillary. Through the open end, the intracellular medium was equilibrated with test solutions and electrical current was injected for the voltage clamp of the membrane in the Tyrode pool. (2.) The capacitive transient on stepping the membrane potential decayed with a time constant of 10-60 microseconds, depending on the capacitive area (20-80 pF). The time course was a single exponential in 46% of the atrial cells and in 66% of the ventricular cells. In these tissues the series resistance, approximated by a ratio of the time constant and Cm, was 686 +/- 180 k omega (n = 37) in the ventricular cells or 812 +/- 143 k omega (n = 18) in the atrial cells. The stable seal resistance (Rseal) established in the oil-gap was around 33 M omega in the ventricular cells and 100 M omega in the atrial cells. (3.) A rapid increase in the inward current followed by a slow decay was observed on repolarization over the range negative to the potassium equilibrium potential. From the inward rectification of both peak and late currents and suppressive effects of Cs+ on the current, the current changes were attributed to activation and inactivation of the inward rectifier K channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Patch-voltage-clamp method for measuring fast inward current in single rat heart muscle cells.

A patch-voltage-clamp method was used to measure fast inward ionic currents in single heart muscle cells. 1. Theoretical analysis including computer simulation has shown that the method provides fast settling of membrane potential (within 10 microseconds) and reliable voltage clamp on the tested membrane patch when the area of the patch is 200-300 times smaller than the area of the whole cell membrane. 2. The transient time at the I-V converter output is increased up to 70-75 microseconds due to the stray capacity in the I-V converter feedback. When fast-response operational amplifiers are used in the set up this transient time may be decreased to 30 microseconds by partial restoration of the high-frequency components of the signal. 3. Experimental data have shown that the ionic channel population in the membrane patch of about 5 micrometers in diameter is on the one hand large enough to directly observe integral ionic current and on the other hand small enough for the fluctuations of ionic current to be appreciable. This permits the method to be applied to ionic current investigations both by classical methods and by statistical analysis.

Animals↗
PubMed1989

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

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

Animals↗
PubMed1980

K efflux through inward rectifying K channels in voltage clamped Purkinje fibers.

The 42K efflux was measured in voltage clamped sheep Purkinje fibers. The voltage dependence of the K efflux can be described as N-shaped, showing a negative slope region. At potentials negative to -30mV, the K efflux is largely due to K flowing through a channel which rectifies in the inward direction and which is blocked by external application of 20mM Cs+. At potentials positive to -30mV an outward rectifier dominates the shape of the K efflux-voltage relationship. This component is insensitive to short external application of Cs+. Both components were also found when Na+ was replaced by tetramethylammonium. When the steady-state current-voltage relationship is compared with the K efflux one can conclude that the outward rectifying K flux largely determines the shape of this curve at positive membrane potentials, while the negative slope region of the K efflux correlates with the negative slope of the steady-state current-voltage relation. The K efflux is only slightly enhanced by stimulation of the preparation, corroborating the finding of inward-going rectification of the K channel. A clamp program repetitively activating the positive dynamic current e.g. by alternating the membrane potential between -70 and +10mV, increases the K efflux by about 50% as compared to the efflux measured in steady-state at this positive membrane potential. 4-Aminopyridine suppresses both this extra K efflux and the positive dynamic current. It is concluded that K ions contribute to the positive dynamic current.

Animals↗
PubMed1987

Stiffness of frog muscle fibres during rise of tension and relaxation in fixed-end or length-clamped tetani.

Stiffness measurements were performed during the rise, the plateau and the relaxation of tetanic contractions both in whole single muscle fibres and in tendon-free fibre segments under either fixed-end or length-clamp conditions. Fibres were isolated from the tibialis anterior muscle of the frog. Experiments were performed at 2-6 degrees C. Changes in length of tendon-free fibre segments were monitored by means of a "striation follower", an opto-electronic device which, during contraction, measured sarcomere displacement at the level of two selected regions of a fibre. Fast length perturbations imposed at one tendon end of a fibre during the plateau of tetanic contractions distribute uniformly along its length. During the tetanus rise stiffness led isometric tension in whole fibres under fixed conditions as well as in tendon-free fibre segments under length-clamp conditions. It was confirmed that a significant part of the unlinearity of T1 relations is determined by tendon compliance. During the isometric phase of relaxation in fixed-end tetani, the decline of tension led that of stiffness both in whole fibres and in tendon-free fibre segments. It is concluded that the shift observed between stiffness and tension during tetanus rise and relaxation represents a true specific event in the contractile process.

Animals↗
PubMed1976

Voltage clamp analysis in isolated cardiac fibres as performed with two different perfusion chambres for double sucrose gap.

Voltage clamp experiments were performed on isolated frog atrial trabeculae disposed in 2 different perfusion chambres for double sucrose gap. In one of the perfusion chambres, a "liquid partition system" (L.P.S.) was used; in the other, a vaseline sealing (V.P.S.) method was used to separate the various fluid compartments. From the linear electrical properties, the elements of an apparent equivalent electrical circuit were calculated. The apparent nodal capacity was significantly larger in fibres disposed in the "liquid partition system" than those disposed in the "vaseline partition system". The apparent "error factor" resulting from the presence of the series resistance was larger in the L.P.S. than in the V.P.S. The apparent "error factor" becomes relatively large when the membrane conductance increases, such as during the flow of the inward current. A rapid desactivation of the peak inward current was found on bringing the command potential back to the resting potential in the V.P.S. This was not found in the L.P.S., indicating better voltage control with the V.P.S. than with the L.P.S. Transmembrane microelectrode recordings during voltage clamp experiment in the V.P.S. indicated satisfactory voltage control during the flow of the peak inward current. Adequate voltage control is lost when notches or irregularities appear on the current traces.

Animals↗
PubMed1981

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.

1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.

Acetylcholine↗
PubMed1990

Ventilatory effects of hypercapnic end-tidal PCO2 clamps during aerobic exercise of varying intensity.

Nine subjects performed a sequence of sustained and randomised changes between 40 W and 100 W on a cycle ergometer while the end-tidal PO2 was kept close to 17.3 kPa (130 mm Hg) by means of a dynamic forcing technique (reference experiment). In a second series inspiratory CO2 was additionally manipulated so as to hold end-tidal PCO2 (PETCO2) near 6.5 kPa (49 mm Hg; 'CO2-clamp' experiment). By this forcing PETCO2 oscillations were attenuated and more evenly distributed over the frequency range. Ventilation (VT) responded to this manoeuvre with an upward trend that could not be ascribed to a slow CO2-response component, changes in metabolic rate or a dissociation of end-tidal and arterial PCO2. VT differences between reference and CO2-clamp experiments were abolished within a 3-min period following the termination of the external CO2 control. The present results suggest that the CO2-H+ stimulus plays a major role in adjusting ventilation when exercise intensity is decreased. The underlying CO2 effect appears to be neither additive nor bi-directionally symmetrical.

Arteries↗
PubMed1990

Haemostatic clips and clamps used in surgical neurology with special reference to non ferro magnetic Phynox spring clips by Aesculap.

This paper concerns the historical developments and technical properties of haemostatic devices used in neurosurgery with the exception of coagulation. Therefore there are described haemostatic clips designed for the scalp, clips used on arteries and veins, a clamp for the reduction of the carotid diameter in the neck, and a clamp for closing the venous dural sinuses. Special reference will be made regarding non ferro magnetic Phynox clips by Aesculap.

Hemostasis, Surgical↗
PubMed1999

The Effect of Nitecapone, a New Antioxidant, on Myocardial Function After Aortic Cross-clamping in Experimental Heart Ischemia.

During aortic cross-clamping, the myocardium suffers from global ischemia, which is followed by reperfusion after declamping. The generation of free oxygen radicals increases during reperfusion, resulting in arrhythmias and impaired cardiac function. This study was conducted to evaluate the effect of a novel antioxidant nitecapone (NC) on cardiac reperfusion injury in vivo. Twelve pigs were anesthetized and after sternotomy the aorta and the right atrium were cannulated for cardiopulmonary bypass. The heart was arrested with either +4 degreesC crystalloid cardioplegia alone in the control group (n = 6) or cardioplegia with NC (50 µM) added in the NC group (n = 6). Cardioplegia was added every 20 minutes. After 1 hour of aortic cross-clamping, blood samples for oxidative stress analysis were taken, and hemodynamic profile surveillance continued for 90 minutes. Heart rate (p = 0.04) and left ventricular end diastolic pressure (LVEDP) (p = 0.04) were significantly lower in the NC group than in the C group after aortic declamping. Cardiac output and myocardial contractility (dP/dtmax) were also enhanced in the group receiving NC, but the difference was not statistically significant. At 30 minutes after reperfusion, the coronary production (coronary sinus-aorta) of thiobarbituric acid reactive substances correlated inversely with cardiac output (r = -0.90, p = 0.001) and stroke volume (r = -0.82, p = 0.007). The effect of NC on lipid peroxidation seems to be modest and therefore the target of NC is unclear. NC would appear, however, to be a beneficial additive in the crystalloid cardioplegia in terms of functional recovery.

Journal Article↗