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.

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A new method is described as an alternative to whole-cell recording in order to prevent "wash-out" of the muscarinic response to acetylcholine (ACh) in rat lacrimal gland cells. The membrane of a cell-attached patch is permeabilized by nystatin in the patch pipette, thus providing electrical continuity between the pipette and the cytoplasm of the cell without the loss or aheration of cytoplasmic compounds necessary for the maintenance of the response to ACh. With normal whole-cell recording in these cells, the response to ACh, seen as the activation of Ca-activated K and CI currents, lasts for ~5 rain. With the nystatin method, the response is not diminished after 1 h. Nystatin, applied extracellularly, is shown to cause a rapid and reversible increase of membrane conductance to cations. In the absence of wash-out, we were able to obtain dose-response curves for the effect of ACh on Ca-activated K currents. An increase of [ACh] caused an increase in the K current, with apparent saturation at concentrations above ~ 1 #M ACh. The delay between ACh application and the activation of K current was inversely related to [ACh] and reached a minimum value of 0.7-1.0 s at high [ACh].

SUMMARY1. Inward currents in chromaffin cells were studied with the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The intracellular solution contained 120 mM-Cs+ and 20 mM-tetraethylammonium (TEA+). Na+ currents were studied after blockade of Ca2+ channels with 1 mM-Co2+ applied externally. Ca2+ currents were recorded after eliminating Na+ currents with tetrodotoxin (TTX). The current recordings were obtained in cell-attached, outside-out and whole-cell recording configurations (Hamill et al. 1981).2. Single channel measurements gave an elementary current amplitude of 1 pA at -10 mV for Na+ channels. This amplitude increased with hyperpolarization between -10 and -40 mV, but did not vary significantly between -40 and -70 mV. 6. At -5 mV, single Ba2+ currents appeared as bursts of 1'9 ms mean duration containing on the average 0-6 short gaps. The burst duration was larger at positive potentials.7.

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, 0. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. (1981) Pflugers Arch. 391, 85-1001, which allows the intracellular Ca2+ concentration to be controlled. With an internal solution highly buffered to low values ofCa2+ 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 ,.M), 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.Exocytosis is used by many cell types to transfer vesicle-contained substances such as neurotransmitters (1), hormones (2), or enzymes (3) into the extracellular medium. A comparatively well-studied example of this process is the secretion of catecholamines in chromaffin cells of the adrenal medulla, where Ca2" entry triggers a cycle involving exocytosis of chromaffin granules (4-6), followed by retrieval ofmembrane from the surface into intracellular vesicles and. vacuoles (5-8). Fusion of chromaffin granules with the plasma membrane leads to a transient increase of membrane surface area, soon followed by a surface decrease corresponding to endocytosis. Such changes should be reflected by alterations of the cell membrane capacitance after stimulation.-The patch-clamp method constitutes a very sensitive way of measuring membrane currents (9), which, with appropriate voltage command signals, can be used to measure membrane capacitance at high resolution. Here, we show that a sensitivity can be reached that should be sufficient to resolve changes in capacitance expected during exocytosis/ endocytosis of single vesicles. Furthermore, we report the observation of discrete changes in the range of 0.4-80 fF (1 fF = 10-i5 F) that have many of the properties expected for such events. METHODSA simple way of measuring the capacitance of a cell membrane is to apply, under voltage-clamp conditions, a sine wave to the membrane and to measure current. At high enough frequencies, the resulting sinusoidal current is roughly proportional to membrane capacitance C, apart from e...

SUMMARY1. Bovine chromaffin cells were enzymatically isolated and kept in short term tissue culture. Their electrical properties were studied using recent advances of the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981).2. When a patch pipette was sealed tightly to a chromaffin cell ('cell-attached configuration') current wave forms due to intracellular action potentials could be observed. The frequency of the wave forms was altered by changing the pipette potential. When acetylcholine was present in the pipette solution, acetylcholineinduced single channel currents were evident in the patch recording. Action potential wave forms were then often seen to follow acetycholine-induced single channel currents.3. In the cell-attached configuration, large single channel current events did not resemble square pulses but showed exponential relaxations with time constants of the order of 50 ms.4. After rupture of the patch of membrane, the pipette-cell seal remained stable ('whole-cell recording ', Hamill et al. 1981). Chromaffin cells were found to have a resting potential of -50 to -80 mV, and an input resistance around 5 GQ. The high cell resistance accounts for the relaxing currents evident in the cell-attached configuration.5. In the best cases, the effective time constant ofthe voltage clamp in the whole-cell recording mode was 15 ps. Exchange of small ions such as Na+ ions between pipette and cell interior solutions was then complete within 15 s. 6. Acetylcholine-induced currents were obtained at various acetylcholine concentrations. Single acetylcholine-induced channels had a slope conductance of 44 pS between -100 and -55 mV, and a mean duration of 27 ms at -80 mV (at room temperature).

No abstract

articlesThe building block of synaptic transmission is the quantum, the minimal increment of postsynaptic signals 1 . At vertebrate neuromuscular junctions, the quantum may be equated with spontaneous signals obtained in the absence of presynaptic action potentials, called miniature currents (or potentials) and believed to be due to release of one neurotransmitter vesicle. For central synapses, this issue remains an open question, as large miniature currents are suggested to arise from the concerted release of several presynaptic vesicles and to be the sum of several quanta 2-5 . Such multivesicular miniature events could reflect tetrodotoxinresistant action potentials in presynaptic terminals 6 . Another explanation comes from evidence for functional intracellular Ca 2+ stores in presynaptic terminals. First, inositoltrisphosphate (InsP 3 ) receptors are immunolocalized in presynaptic terminals of the deep cerebellar nuclei and retina 7,8 . Second, at the frog neuromuscular junction, agents that affect ryanodine-sensitive Ca 2+ stores also regulate presynaptic intracellular Ca 2+ (Ca 2+ i ) rises and acetylcholine release during high-frequency stimulation 9,10 . Third, action-potential-evoked release of acetylcholine at synapses in Aplysia buccal ganglia is inhibited by ryanodine and augmented by presynaptic injection of cyclic ADP ribose 11 . Fourth, caffeine and/or ryanodine modify presynaptic Ca 2+ i signals in autonomic ganglia 12,13 and in photoreceptors 14 . Finally, in hippocampal pyramidal cells, caffeine or thapsigargin can increase the frequency of miniature IPSCs 15 . Hence, spontaneous Ca 2+ release from presynaptic Ca 2+ stores may provide the synchronization mechanism that leads to multivesicular miniatures. However, except for one study that gave negative results in cultured retinal amacrine cells 16 , this possibility has not been tested systematically.To assess the contribution of intracellular Ca 2+ stores to neurotransmitter release, we monitored the amplitude distribution of miniature synaptic currents while manipulating potential presynaptic Ca 2+ stores. Using cerebellar interneuron-Purkinje cell synapses, in which large miniature synaptic currents are prominent, we found that the largest mIPSCs result from multivesicular release and depend on Ca 2+ mobilization from ryanodine-sensitive presynaptic stores. Further, two-photon confocal microscopy showed ryanodine-sensitive intracellular Ca 2+ i transients highly localized to presumed release sites, which may underlie large miniature currents. RESULTSMiniature IPSCs recorded in cerebellar Purkinje cells, at -60 mV under symmetrical Cl -concentrations and in the presence of tetrodotoxin (TTX) and ionotropic glutamate receptor blockers, showed mean amplitudes of 125 ± 9 pA (n = 28 cells), larger than for most neurons (Fig. 1a). Amplitude histograms had a distinct peak for values less than 200 pA, followed by a long tail with amplitudes up to 1500 pA (Fig. 1b). Spurious summation of independent events did not contribute to the generation of lar...