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At least 1,567 records · Page 87Linked to original sources
PubMed1991

High-resolution measurement and calibration of Ca(2+)-transients using Indo-1 in guinea-pig atrial myocytes under voltage clamp.

Spherical atrial myocytes obtained by enzymatic dispersion of hearts from adult guinea-pigs were loaded with the fluorescent Ca(2+)-indicator Indo-1 via patch-clamp pipettes. The dialysing solution additionally contained citrate (60 mM) as low-affinity ('linear') Ca(2+)-chelating compound in order to slow intracellular Ca(2+)-transients. Changes in Indo-1 fluorescence under voltage-clamp due to Ca(2+)-entry and/or release from the SR were calibrated using an in vivo procedure to determine the limiting fluorescence ratios. Sample recordings will be presented to demonstrate that components of a [Ca2+]i-transient due to entry via L-type Ca(2+)-channels and due to Ca(2+)-release from the SR can be directly visualized.

Animals↗
PubMedDate not supplied

Ca2+ oscillations induced by histamine H1 receptor stimulation in HeLa cells: Fura-2 and patch clamp analysis.

The response of HeLa cells to histamine H1 receptor stimulation is characterized by periodic increases in cytosolic free Ca2+ concentration. The mechanisms underlying this oscillatory behaviour are not well understood. Fura-2 and patch clamp experiments carried out on HeLa cells have previously shown: (a) that Ca2+ oscillations are not initially dependent on the presence of external Ca2+, that external Ca2+ is required to maintain the oscillatory activity; (b) that a depolarization of the cell membrane leads to an inhibition of Ca2+ oscillations during the external Ca2+ dependent phase of the process; and (c) that Ca2+ oscillations can be abolished during this latter phase by the exogenous addition of Ca2+ channel blocking agents, such as Co2+ or La3+. The contribution of the inositol phosphate pathway to Ca2+ oscillations was more recently investigated in whole cell experiments performed with patch pipettes containing IP3 or the non-hydrolysable GTP analogue GTP-gamma S. Clear periodic current fluctuations were recorded using both patch pipette solutions. Assuming that the intracellular IP3 level remained constant under these conditions, these findings provide direct evidence that the Ca2+ oscillations in HeLa cells do not arise from a periodic production of IP3. The effect of the internal and external cell pH on the oscillatory process was also investigated in Fura-2 and patch clamp experiments. It was found that an increase in intracellular pH from 7.4 to 7.7 during the external Ca2+ dependent phase of the histamine stimulation abolishes the appearance of Ca2+ spikes whereas, a cellular acidification to pH 7.2 maintains or stimulates the Ca2+ oscillatory activity. The former effect was observed in the absence of Ca2+ in the bathing medium, indicating that the inhibitory action of alkaline pH was not related to a reduced Ca2+ entry. An increase in extracellular pH from 7.3 to 9.0 in contrast elicited an intracellular Ca2+ accumulation which resulted in most cases in an inhibition of the oscillatory process. This effect was dependent on external Ca2+ and was observed in alkaline internal pH conditions (pH 7.7). These observations suggest: (a) that the net Ca2+ influx in HeLa cells is strongly dependent on the cell internal and external pH; and (b) that the magnitude of this Ca2+ influx controls to a large extent the oscillation frequency. Finally, an inhibition of the histamine induced Ca2+ oscillatory activity was observed following the addition of the Ca(2+)-induced Ca(2+)-release (CICR) inhibitor adenine to the external medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1991

Physiological and pharmacological properties of 5-HT3 receptors--a patch clamp-study.

Whole cell and patch clamp techniques were used to investigate the properties of 5-HT3 receptors of a murine neuroblastoma cell line (N1E-115) and adult rabbit nodose ganglion neurones. In addition, some preliminary results from guinea-pig nodose ganglion neurones are presented. In such cells, voltage-clamped at -60 mV, 5-HT (10 microM) induced an inward current associated with a conductance increase. The results of ion substitution experiments suggest that the 5-HT activated ion channel is permeable to both Na+ and K+ ions with a permeability ratio (PNa/PK) of 0.94 and 0.92 for rabbit nodose ganglion cells and N1E-115 cells respectively. On outside out membrane patches excised from rabbit nodose ganglion neurones, 5-HT (1 microM) activated clearly discernible single channel currents with a conductance of 16.6 +/- 0.7 pS (n = 4). In contrast, fluctuation analysis of 5-HT induced whole cell currents suggests that the single channel conductance of N1E-115 cells is only 0.3 pS, a value some 50 fold lower. The 5-HT-induced whole cell currents recorded from all three preparations were antagonised by the selective 5-HT3 receptor antagonist ondansetron (GR38032F) and by the less selective agents metoclopramide, cocaine and (+)-tubocurarine. However, these preparations demonstrate a differential sensitivity to some antagonists. In particular, (+)-tubocurarine was a potent antagonist in N1E-115 cells (IC50 = 0.85 nM) but was approximately 200 fold (IC50 = 156 nM) and 1200 fold (IC50 = 10 microM) less potent in rabbit and guinea-pig nodose ganglion neurones respectively. Additionally, a novel effect of ketamine (10 microM) to potentiate the 5-HT-induced current of rabbit nodose ganglion neurones is described.

Action Potentials↗
PubMed1983

A shielded microelectrode suitable for single-electrode voltage clamping of neurones in the CNS.

The fabrication of a shielded microelectrode is described which has a time constant of about 3 microseconds, regardless of its depth in neural tissue. The shield is insulated from surrounding tissue with an insulation resistance which exceeds 1000 M omega, and the insulation will withstand +/- 15 V. Because of its narrow profile, the electrode is suitable for in vivo intracellular recordings from motoneurones and other large neurones of the CNS. The small time constant and the high breakdown resistance of the electrode make it suitable for use with single-electrode voltage clamp circuitry to voltage clamp motoneurone somata using switching rates in excess of 30 kHz.

Animals↗
PubMed1987

A preparation for patch clamp studies of labelled, identified neurones from guinea pig spinal cord.

Details are described of techniques which allow the isolation of labelled, identified neurones suitable for patch clamp recording from the guinea pig spinal cord. Fluorescent labels, injected into either the hindlimb muscles or the cerebellum, are retrogradely transported to motoneurones or dorsal spinocerebellar tract neurones respectively. Cells are then enzymatically dissociated from spinal cord slices and identified using fluorescence microscopy. Patch clamp or whole cell recordings are then made.

Afferent Pathways↗
PubMed1988

Spatial control of membrane potential: a method for improved voltage clamping of the squid giant synapse.

In voltage clamp experiments at the squid giant synapse anisopotentiality of the presynaptic terminal can distort measurements of synaptic transfer. Local compression of the presynaptic cell, at the point where the terminal joins its axon, appears to reduce this problem by electrically isolating the terminal from the axon. Because compression reduced anisopotentiality in the presynaptic terminal and altered the apparent voltage dependency of transmitter release, this procedure may be a simple way to improve voltage clamp control in this and other long cells. Compression of the presynaptic cell also reduced the diffusion of injected molecules out of the terminal region. This increased the effective concentration of given quantities of tetraethylammonium and Fast green injected into the presynaptic terminal. Thus, compression should also facilitate experiments in which molecules are microinjected into this and other cells because the effective volume of the cell is reduced and less of the injected substance will be required.

Animals↗
PubMed1990

A pipette holder for use in patch-clamp measurements.

A novel design for a pipette holder for use in patch-clamp experiments is presented. The holder is designed for use in patch-clamp amplifiers equipped with female BNC-type connectors on the headstage. In this design the glass micropipette cannot contact the Ag/AgCl electrode, thus avoiding deterioration and subsequent offset voltages and baseline current drifts of the Ag/AgCl electrode, induced by frequent replacement of micropipettes.

Electrophysiology↗
PubMed1991

A simple and multi-purpose "concentration-clamp" method for rapid superfusion.

A simple method for the rapid exchange of the medium used for a variety of electrophysiological experiments including patch-clamp experiments on cultured cells is described. The advantages of this method over the conventional ones are that this solution exchange system has a very simple structure and thus is inexpensive and can be easily installed in already existing experimental setups. The method involves the use of a fine double-barrelled polyethylene needle placed near the cell or other isolated preparations under recording. Electromagnetic valves are used to switch the solutions in the polyethylene tubes which enable a rapid and localized application of test solutions as well as their rapid washout. The latency of solution exchange, as measured by a change in the holding current under voltage-clamp experiments was fairly rapid (about 120 ms). Neither was the recording interrupted nor the sealing resistance affected during repeated exposure to test solutions. This method is proving to be useful, particularly for electrophysiological and pharmacological studies on immovable cells such as those in culture.

Animals↗
PubMed1991

Room temperature culture extends the useful life of adult neurons for voltage-clamp experiments.

Isolated superior cervical ganglion (SCG) neurons were maintained in vitro at 22 degrees C (22 degrees C neurons) for up to 4 days in an effort to inhibit process outgrowth and thus extend the useful life of SCG neurons for voltage-clamp experiments. The neurons were viable after 4 days in vitro and remained roughly spherical whereas neurons maintained in vitro at 37 degrees C (37 degrees C neurons) developed extensive neurite processes after 2 days. The resting potential of 22 degrees C neurons was more hyperpolarized and the action potential duration was reduced compared to acutely isolated neurons (acute neurons) or 37 degrees C neurons. The steady state Na+ current activation and inactivation parameters of the acute and 22 degrees C neurons were similar whereas the half activation voltage was hyperpolarized and the slope factor of inactivation was reduced for the 37 degrees C neurons compared to the other two groups. The Na+ currents recorded from the 37 degrees C neurons displayed obvious signs of poor voltage control which were not observed in the acute or 22 degrees C neurons. The acetylcholine (ACh) sensitivity of both 22 degrees C and 37 degrees C neurons was significantly less than that of the acute neurons. This report demonstrates that room temperature culture of SCG neurons is a simple method which prevents process outgrowth and thus extends the useful life of the neurons for voltage-clamp experiments.

Acetylcholine↗
PubMed1993

Oxygen tension clamp around single neurons in vitro: a computerized method for studies on O2 deprivation.

This report presents a novel computerized oxygen tension clamp for use with single neurons. One main objective for developing such a system is to be able to study the intrinsic response of single neurons to graded hypoxia. The device described in this paper is comprised of three major subsystems: (1) an oxygen monitoring subsystem, (2) a motorized perfusion subsystem and (3) a computer subsystem for data acquisition and control. The clamp uses an oxygen scavenger (sodium hydrosulfite) to decrease O2 availability. The system has many advantageous features: (a) studies on hypoxia can be carried out without using chemical metabolic inhibitors to simulate hypoxia; (b) precise control of the O2 level around a given cell is feasible for extended periods of time; (c) rapid and accurate changes in the O2 level around a cell can be made because the O2 level is continuously monitored and adjusted by a computer; (d) other cells outside of the immediate vicinity of the cell being studied are not subjected to hypoxic exposures; and (e) the system is inexpensively built around readily available components.

Cell Separation↗
PubMed1993

The dynamic clamp: artificial conductances in biological neurons.

The dynamic clamp is a novel method that uses computer simulation to introduce conductances into biological neurons. This method can be used to study the role of various conductances in shaping the activity of single neurons, or neurons within networks. The dynamic clamp can also be used to form circuits from previously unconnected neurons. This approach makes computer simulation an interactive experimental tool, and will be useful in many applications where the role of synaptic strengths and intrinsic properties in neuronal and network dynamics is of interest.

Animals↗
PubMed1989

Ionic currents in the soma of an identified cockroach motoneurone recorded under voltage-clamp.

1. Membrane currents have been recorded from the soma of a bifunctional basalar/coxal depressor motoneurone in the metathoracic ganglion of the cockroach (Periplaneta americana) using a two-electrode voltage-clamp technique. 2. This motoneurone cell body is normally inexcitable when studied under current-clamp. Appropriate depolarizing command steps evoke rapid transient outward currents and late outward currents. 3. Late outward currents are dominated by a Ca-dependent component that confers an N-shaped I-V relationship on the neurone. 4. The Ca-dependent outward current is suppressed by Cd2+ (1 mM), Mn2+ (5 mM) or verapamil (50 microM). 5. Externally applied tetraethylammonium ions (TEA+) (25 mM) block the Ca-dependent current, but also appear to suppress a component of the late outward current that is independent of Ca2+. 6. Aminopyridines cause only minor suppression of late outward currents, but shift the peak in the N-shaped I-V relationship to more negative potentials. 7. The reversal potential of tail currents recorded following pre-pulses to +50 mV were dependent upon the pre-pulse duration; increasing the duration from 10 to 50 msec caused a +17 mV shift in tail current reversal potential. 8. A five-fold increase in the K+ concentration of the solution bathing the preparation only produced small and inconsistent changes in the reversal potential of tail currents. 9. Five-fold reduction in external Cl- caused no change. 10. The dependence of tail current reversal potential upon pre-pulse duration and the limited effect of alterations in the composition of the bathing solution are discussed in the context of restricted ion movements near the external surface of the cell membrane.

Aminopyridines↗
PubMed1983

M-current in voltage-clamped olfactory cortex neurones.

The soma of olfactory cortex neurones in vitro was voltage-clamped by means of a single microelectrode sample-and-hold technique. In most neurones, hyperpolarizing voltage commands from relatively positive holding potentials (-40 to -50 mV) elicited a slow inward current relaxation with voltage-dependent and kinetic properties similar to the non-inactivating K+-current (M-current; IM), first described in amphibian sympathetic neurons. Deactivation of IM at negative potentials probably accounts for the slow sag of the hyperpolarizing electrotonic potential measured during current-clamp experiments. IM was inhibited by the cholinergic agonist muscarine or barium ions.

Animals↗
PubMed1990

Lesion patterns of vasoactive intestinal polypeptide-containing neurons in the myenteric plexus induced by clamping or transsection of rat jejunum.

The acute reactions of vasoactive intestinal polypeptide (VIP)-containing neurons in the myenteric plexus of the rat jejunum in response to circumferential clamping or transection/reanastomosis were evaluated in a detailed time course of up to ten days. Whereas in sham-operated controls VIP immunostaining was confined exclusively to varicose fibers, either clamping or transection induced the appearance of strongly VIP-immunoreactive beaded neuronal processes and neuronal perikarya in the oral part of the lesion one day postoperatively. After five days strongly immunostained varicose fibers orientated in the longitudinal axis of the gut towards the lesion reappeared suggestive of the onset of regenerative processes.

Animals↗
PubMed1991

Whole-cell patch clamp recordings from rhythmically active motoneurons in the isolated spinal cord of the chick embryo.

Whole-cell patch clamp recordings were obtained during motor activity from electrically identified motoneurons within the spinal cord of the chick embryo maintained in vitro. Most recordings were performed on E11-E13 motoneurons although it was also possible to record from younger cells (E7-E9). Voltage clamp recordings were used to characterize the synaptic currents expressed in femoro-tibialis (extensor) motoneurons during motor activity. These motoneurons exhibited rhythmic excitatory currents with reversal potentials near 0 mV. This powerful technique enables high resolution recordings from identified motoneurons in situ and allows investigation of the membrane and synaptic mechanisms involved in the development of embryonic motility.

Animals↗
PubMed1991

Direct identification of individual vasopressin-containing nerve terminals of the rat neurohypophysis after 'whole-cell' patch-clamp recordings.

The membrane currents in rat neurophypophysial nerve terminals, which contain either vasopressin or oxytocin, have been previously recorded using the 'whole-cell' patch-clamp technique. Interpretation of the electrophysiological data would be significantly strengthened by the ability to correlate them with knowledge of the peptide contents of the terminals being studied. Here, a novel method for detection of the peptide hormone, arginine vasopressin, in those individual isolated terminals is described. The unique aspect of this procedure is that the contents of the terminal are aspirated into the recording electrode after 'whole-cell' patch-clamp recording, and then a highly sensitive dot immunobinding assay allows identification of the peptide contents in the terminals.

Animals↗
PubMed1992

The nature of the acetylcholine receptor in a Buccinum proboscis muscle examined by the sucrose-gap voltage clamp technique.

1. ACh dose-response curves for the radicular retractor muscle of Buccinum showed maximum force and membrane depolarisation of 3.3 mV at 50 mumol l-1 ACh. 2. PCh was found to be almost a full agonist for force and induced higher membrane depolarisations than ACh while BCh was only a partial agonist of very low potency. This suggests an AChR neither muscarinic nor nicotinic in mammalian terminology. 3. Neither muscarine nor nicotine had any direct agonistic effects on the muscle but pre-exposure to nicotine inhibited both force and membrane depolarisation induced by a subsequent dose of ACh. 4. The specific muscarinic and nicotinic antagonists atropine, d-tubocurarine and gallamine all inhibited ACh responses in a dose-dependent manner. 5. Single sucrose-gap recording showed that ACh induced a depolarisation resulting in a contracture. Double sucrose-gap voltage clamp recording showed that 10 mumol l-1 ACh induced an inward transmembrane current of ca 2 microA. Both ACh-induced depolarisation and inward current were abolished in Na-free media. 6. When clamped at a series of membrane voltages between natural Em and positive potentials the ACh-induced Na-dependent inward current declined as Em was reduced and was abolished at -10 mV. This current showed no reversal even at strong positive membrane voltages. 7. The AChR of this muscle appears to be neither exclusively nicotinic nor muscarinic but a hybrid and shows characteristics of voltage inactivation.

Animals↗
PubMed1983

Sodium and calcium currents measured in isolated catfish horizontal cells under voltage clamp.

A low resistance suction microelectrode was used to record intracellularly from enzymatically dissociated horizontal cells obtained from catfish retina (Ictalurus punctatus). This microelectrode was connected to a voltage clamp circuit and the transmembrane currents were recorded during depolarizing and hyperpolarizing clamp pulses. Two fast transient inward currents were recorded, one sensitive to tetrodotoxin and one insensitive to this drug. A slower and persistent tetrodotoxin insensitive inward current was also recorded. The results indicate that separate membrane conductances exist for both sodium and calcium ions. We suggest that the action potential recorded from isolated horizontal cells is generated by both sodium and calcium currents.

Animals↗