Patch-clamp techniques for time-resolved capacitance measurements in single cells.
01 Feb 1988-Pflügers Archiv: European Journal of Physiology (Pflugers Arch)-Vol. 411, Iss: 2, pp 137-146
TL;DR: 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 by using a time domain technique and a lock-in amplifier.
Abstract: 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. Particurlarly, it is equally applicable to the “slow whole-cell” configuration (access resistance in the range 100 MΩ to 1 GΩ) and to normal whole-cell measurements (access resistance typically 10 MΩ). 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.
Citations
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TL;DR: In this article, the authors show that under voltage-clamp conditions, stochastically occurring signals can be recorded from adrenal chromaffin cells using a carbon-fibre electrode as an electrochemical detector.
Abstract: In synapses, a rise in presynaptic intracellular calcium leads to secretory vesicle fusion in less than a millisecond, as indicated by the short delay from excitation to postsynaptic signal. In nonsynaptic secretory cells, studies at high time resolution have been limited by the lack of a detector as fast and sensitive as the postsynaptic membrane. Electrochemical methods may be sensitive enough to detect catecholamines released from single vesicles. Here, we show that under voltage-clamp conditions, stochastically occurring signals can be recorded from adrenal chromaffin cells using a carbon-fibre electrode as an electrochemical detector. These signals obey statistics characteristic for quantal release; however, in contrast to neuronal transmitter release, secretion occurs with a significant delay after short step depolarizations. Furthermore, we identify a pedestal or 'foot' at the onset of unitary events which may represent the slow leak of catecholamine molecules out of a narrow 'fusion pore' before the pore dilates for complete exocytosis.
805 citations
TL;DR: A given synaptic vesicle can exocytose with high probability within a few hundred microseconds, if [Ca2+]i rises above lOOµM, and these properties provide for the extremely rapid signalling required for neuronal communication.
Abstract: Rapid calcium-dependent exocytosis underlies neurotransmitter release from nerve terminals. Despite the fundamental importance of this process, neither the relationship between presynaptic intracellular calcium ion concentration ([Ca2+]i) and rate of exocytosis, nor the maximal rate of secretion is known quantitatively. To provide this information, we have used flash photolysis of caged Ca2+ to elevate [Ca2+]i rapidly and uniformly in synaptic terminals, while measuring membrane capacitance as an index of exocytosis and monitoring [Ca2+]i with a Ca(2+)-indicator dye. When [Ca2+]i was abruptly increased to > 10 microM, capacitance rose at a rate that increased steeply with [Ca2+]i. The steepness suggested that at least four calcium ions must bind to activate synaptic vesicle fusion. Half-saturation was at 194 microM, and the maximal rate constant was 2,000-3,000 s-1. A given synaptic vesicle can exocytose with high probability within a few hundred microseconds, if [Ca2+]i rises above 100 microM. These properties provide for the extremely rapid signalling required for neuronal communication.
729 citations
TL;DR: Fluorescent compounds and specific ion currents were used to study the kinetics of the diffusional exchange between small cells and patch pipettes in the tight seal whole cell configuration of the patch clamp technique and diffusion time constants were linearly related to the access resistance.
Abstract: (1) Fluorescent compounds and specific ion currents (Na+, K+) were used to study the kinetics of the diffusional exchange between small cells and patch pipettes in the tight seal whole cell configuration of the patch clamp technique. (2) Changes in the intracellular concentration of the test substances following patch rupture could be fitted with single exponentials, provided the access resistanceR
A of the pipette remained constant during diffusional equilibration. The diffusion time constants were linearly related to the access resistance. (3) When apparent diffusion rates were normalized with respect to access resistance they were found to be dependent on the cell size. However, the cell capacitance, which is proportional to the membrane area turned out not to be a precise measure of the cell size. (4) The experimental diffusion rates changed systematically with the aqueous diffusion coefficient and the inverse third root of the molecular weight. Linear interpolation with respect to these quantities provided estimates of diffusion time constants for the diffusion between patch pipettes and the cytoplasm for substances of interest.
645 citations
TL;DR: A review of animal models of the epilepsies can be found in this paper, where the authors present a review of 50 models for simple partial seizures, including topical convulsants, acute electrical stimulation, cortically implanted metals, cryogenic injury, and complex partial seizures.
618 citations
TL;DR: The caged calcium compound DM-nitrophen is used to investigate the kinetics of calcium-dependent secretion in bovine chromaffin cells and both secretion and movement of vesicles between pools appear to be [Ca2+]i sensitive.
516 citations
References
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TL;DR: 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.
Abstract: 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.
17,136 citations
"Patch-clamp techniques for time-res..." refers background or methods in this paper
...Fast-whole-cell recording. In patch-clamp experiments, the event of patch disruption leading to the whole-cell configuration is indicated by the appearance of large capacitive current transients in response to voltage pulses ( Hamill et al. 1981 )....
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...Standard patch clamp techniques were employed as described by Hamill et al. (1981) ....
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1,146 citations
"Patch-clamp techniques for time-res..." refers methods in this paper
...This type of amplifier has in the past been used extensively in circuit analysis and in spectroscopy (Horowitz and Hill 1980)....
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TL;DR: The size distribution of step-like capacitance changes is well compatible with the idea that steps of capacitance increase reflect individual events of exocytosis of chromaffin granules, whereas steps of the opposite polarity reflect the formation of vesicles or vacuoles by endocytotic processes.
Abstract: The capacitance of the surface membrane of small adrenal chromaffin cells was measured with patch-clamp pipettes. Continuous and discrete changes of capacitance were observed. They were interpreted as changes of surface area connected to exocytotic or endocytotic processes. Most of the measurements were performed in the "whole-cell" recording configuration [Hamill, O. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. (1981) Pflugers Arch. 391, 85-100], which allows the intracellular Ca2+ concentration to be controlled. With an internal solution highly buffered to low values of Ca2+ concentration (10 nM), the surface capacitance usually decreased and could not be markedly changed by electrical stimulation. At low buffering capacity and medium Ca2+ concentrations (0.1-1 microM), the capacitance measurement showed large fluctuations and discrete steps, reflecting both capacitance decrease and increase. A large transient increase of capacitance could be induced by electrical stimulation under these conditions. It was linked to Ca2+ currents through the membrane. Relatively large (2-6 x 10(-14) F) steps of capacitance decrease were common after extensive stimulation. The size distribution of step-like capacitance changes is well compatible with the idea that steps of capacitance increase reflect individual events of exocytosis of chromaffin granules, whereas steps of the opposite polarity reflect the formation of vesicles or vacuoles by endocytosis.
971 citations
"Patch-clamp techniques for time-res..." refers background or methods in this paper
...The fusion of single granules with the plasma membrane typically results in a capacitance increase of 1 0 - 20 fF (Neher and Marty 1982; Fernandez et al. 1984)....
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...Discrete changes in capacitance related to the exocytosis of single secretory vesicles were recorded with this technique (Neher and Marty 1982; Fernandez et al. 1984)....
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01 Jan 1983
TL;DR: It is the experience that this way of penetrating a cell inflicts much less damage on the cell than the standard microelectrode impalement.
Abstract: Although patch pipettes were originally developed for the recording of single channels, they can be of great advantage for more conventional recordings from whole cells, particularly when the cells under study are small. As described by Hamill et al. (1981), a membrane patch can be broken by a short pulse of suction after a gigaseal has been formed. Very often, this manipulation does not damage the pipette-membrane seal, so that a conductive pathway is established between the pipette and the cell interior, which is well insulated from the bath. It is our experience that this way of penetrating a cell inflicts much less damage on the cell than the standard microelectrode impalement.
730 citations
"Patch-clamp techniques for time-res..." refers background in this paper
...In a whole-cell recording the dominant noise source above 200- 500 Hz is that of GA and CM in series ( Marty and Neher 1983 )....
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...At lower frequencies other noise sources become dominant ( Marty and Neher 1983 )....
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...However, they are useful for the study of cells with simple geometry such as most small secretory cells, which can be accurately described by a three component network ( Marty and Neher 1983 )....
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...The access resistance RA ranges from 2 to 20 Mf~ for typical patch pipettes ( Marty and Neher 1983 )....
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Book•
01 Jan 1980
TL;DR: The Art of Electronics as mentioned in this paper is widely accepted as the best single authoritative book on electronic circuit design and includes 90 oscilloscope screenshots illustrating the behavior of working circuits, dozens of graphs giving highly useful measured data of the sort that is often buried or omitted in datasheets but which you need when designing circuits, and 80 tables (listing some 1650 active components), enabling intelligent choice of circuit components by listing essential characteristics (both specified and measured) of available parts.
Abstract: At long last, here is the thoroughly revised and updated third edition of the hugely successful The Art of Electronics. It is widely accepted as the best single authoritative book on electronic circuit design. In addition to new or enhanced coverage of many topics, the third edition includes 90 oscilloscope screenshots illustrating the behavior of working circuits, dozens of graphs giving highly useful measured data of the sort that is often buried or omitted in datasheets but which you need when designing circuits, and 80 tables (listing some 1650 active components), enabling intelligent choice of circuit components by listing essential characteristics (both specified and measured) of available parts. The new Art of Electronics retains the feeling of informality and easy access that helped make the earlier editions so successful and popular. It is an indispensable reference and the gold standard for anyone, student or researcher, professional or amateur, who works with electronic circuits.
712 citations