Electrical development in spinal cord cell culture

Jackson, MB; Lecar, Harold; Brenneman, DE; Fitzgerald, Sandra C.; Nelson, P. G.

doi:10.1523/jneurosci.02-08-01052.1982

Cited by 65 publications

(31 citation statements)

References 22 publications

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“…For each opening the time between the point at which the channel opened above one-fourth of the full open channel amplitude and the point at which it dropped below this level were recorded. These times were subsequently corrected to correspond to the lifetime of idealized full-amplitude current pulses that would give the same one-fourth amplitude duration when injected into the patch-clamp headstage amplifier (24) (24) and for acetylcholine receptors on several species of cultured muscle cells (25,26). Two exponentials could result from two different open molecular states of a single population of acetylcholine receptors with indistinguishable conductance, from two populations of acetylcholine receptors with the same conductance, or from more complicated kinetic schemes.…”

Section: Methodsmentioning

confidence: 99%

Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor.

Tank¹,

Huganir²,

Greengard³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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Small unilamellar vesicles containing purified and reconstituted nicotinic acetylcholine receptors from Torpedo electroplax have been fused by a simple freeze-thaw procedure to form large liposomes. Giga-seal patch-recording techniques were used to form isolated patches of liposome-membrane and to measure single-channel properties of the reconstituted receptor-ion channel complex. The observed properties are quantitatively similar to those reported for vertebrate muscle nicotinic acetylcholine receptor species recorded in situ. The results demonstrate that the pentameric complex consisting of the q218ft subunits is fully functional. The methods used in these experiments should be useful in studying the effects of chemical alterations on the properties of acetylcholine receptor channels as well as other types of purified and reconstituted ion channels.The nicotinic acetylcholine receptor from Torpedo electroplax is a chemically gated ion channel consisting of a pentameric complex of the five subunits a2fy8S (for reviews, see refs. 1 and 2). It is reproducibly purified and, when reconstituted into small liposomes or planar lipid bilayers, it exhibits the basic agonistinduced cation transport properties anticipated from electrophysiological measurements in vivo (3-10). By applying recently developed methods (11, 12) for patch-recording from large liposomes containing reconstituted ion channels, we have now measured, with high resolution, single-channel properties of the purified and reconstituted Torpedo acetylcholine receptor. We report here that this pentameric complex showed an agonist-independent main conductance level, a subconductance state, saturation of the single-channel conductance at high Na+ concentration, slow inactivation and burst-kinetics at desensitizing concentrations of agonists, and open channel lifetime distributions that are not described by single exponentials. The similarity of these properties with those reported for other vertebrate acetylcholine receptor species recorded in situ provides further evidence that the purified receptor is fully functional and that the well-characterized biochemical properties associated with this isolated receptor complex may apply to a wide class of nicotinic receptor ion channels. MATERIALS AND METHODSPreparation of Liposomes Containing Reconstituted Acetylcholine Receptor. Acetylcholine receptor was isolated, purified, and reconstituted as described (4). Briefly, postsynaptic membranes rich in acetylcholine receptor were prepared from freshly dissected electric organ of Torpedo Californica. The receptor was then purified from a Na cholate extract of the postsynaptic membranes by affinity chromatography on a choline carboxymethyl affinity gel as described by Huganir and Racker (4), except that the loaded column was washed with 20 column. vol of "wash buffer" before elution. The purified and solubilized receptor was incorporated into asolectin (Associated Concentrates, Woodside, NY) small unilamellar vesicles by the detergent dialysis technique. The ...

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Section: Methodsmentioning

confidence: 99%

Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor.

Tank¹,

Huganir²,

Greengard³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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“…In large neurons some of the terminal regions have discernable and identifiable boutons. Other laboratories have shown that these morphologically identifiable connections or appositions are correlated with a high degree of synaptic activity (28,42), In addition, electrophysiological recordings from these regions have shown that the evoked excitatory postsynaptic potentials approximate a normal distribution with no evidence of preferred amplitudes as might occur with relatively low mean quantum content (28,42). Of course at our level of morphology and microscopic resolution we cannot draw any definite conclusions as to the functional properties of each of these terminal structures, but for FPR measurements and for physiological exploration of cells regions, the visualization by phase or differential interference contrast microscopy provides a useful guide.…”

Section: Characteristics Of Ceu Culturesmentioning

confidence: 96%

“…Cultures were studied after 2-6 wk of development in vitro at which time functional synaptic connections measured electrophysiologically (28,42), the maturation of neurons, and the densities of sodium channels assessed by neurotoxin binding, were at a maximum. The stage of neuronal growth was determined by immunocytochemistry for the appearance and locale of each of the neurofilament proteins.…”

Section: Preparation and Labeling Of Cellsmentioning

confidence: 99%

Distribution and lateral mobility of voltage-dependent sodium channels in neurons [published erratum appears in J Cell Biol 1989 May;108(5):preceding 2001]

Angelides

Elmer

Loftus

et al. 1988

The Journal of Cell Biology

146

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Abstract. Voltage-dependent sodium channels are distributed nonuniformly over the surface of nerve cells and are localized to morphologically distinct regions. Fluorescent neurotoxin probes specific for the voltagedependent sodium channel stain the axon hillock 5-10 times more intensely than the cell body and show punctate fluorescence confined to the axon hillock which can be compared with the more diffuse and uniform labeling in the cell body. Using fluorescence photobleaching recovery (FPR) we measured the lateral mobility of voltage-dependent sodium channels over specific regions of the neuron. Nearly all sodium channels labeled with specific neurotoxins are free to diffuse within the cell body with lateral diffusion coefficients on the order of 10 -9 cm2/s. In contrast, lateral diffusion of sodium channels in the axon hillock is restricted, apparently in two different ways. Not only do sodium channels in these regions diffuse more slowly (10-1°-10 -H cm2/s), but also they are prevented from diffusing between axon hillock and cell body. No regionalization or differential mobilities were observed, however, for either tetramethylrhodaminephosphatidylethanolamine, a probe of lipid diffusion, or FITC-succinyl concanavalin A, a probe for glycoproteins. During the maturation of the neuron, the plasma membrane differentiates and segregates voltagedependent sodium channels into local compartments and maintains this localization perhaps either by direct cytoskeletal attachments or by a selective barrier to channel diffusion.

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“…In these cultures, the morphologies and stages of neuronal maturation resemble those described for several types of cultured neurons (e.g., hippocampal neurons: Bartlett andBanker, 1984, Dotti et al, 1988;or striatal neurons: Weiss et al, 1986). In similar cultures, functional glycinergic synapses are detected by patch-clamp recording from the 5th day in vitro (Jackson et al, 1982). At this stage, synaptophysin is detected inside neuritic varicosities contacting neurons, and GlyR forms clusters at the surface of cultured neurons .…”

Section: Characteristics Of Culturesmentioning

confidence: 99%

Gephyrin accumulates at specific plasmalemma loci during neuronal maturation in vitro

1996

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The distribution of glycine receptor (GlyR)-associated gephyrin has been investigated in rat spinal cord neurons maintained in vitro by means of immunocytochemical techniques. Gephyrin, which is crucial for the stabilization of postsynaptic GlyR microdomains, is present in mature neurons at postsynaptic differentiations. With immunofluorescence, discontinuous patches of gephyrin were detected within the neuronal soma of spinal cord neurons on the 1st day after plating. Subsequently, gephyrin was present at membrane areas that correspond to points of contact between cells or with the culture dish. By the 5th day, gephrin was mostly associated with the MAP2-positive somatodendritic compartment. With immunoelectron microscopy, gephyrin blobs detected at the earliest stages (1-3 days after plating) were found within the cytoplasm or associated with the plasma membrane. Asymmetrically immunostained intercellular contacts were only detected after 5 days, and gephyrin was found in association with clearly differentiated postsynaptic membranes at 7 days. At later stages, we observed gephyrin immunoreactivity only at some synapses. Our results suggest that gephyrin accumulates initially at the locus of cell-to-cell contacts involved in adhesion processes. These localizations may define hot spots for later accumulation of the GlyR and possibly other receptors.

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Electrical development in spinal cord cell culture

Cited by 65 publications

References 22 publications

Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor.

Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor.

Distribution and lateral mobility of voltage-dependent sodium channels in neurons [published erratum appears in J Cell Biol 1989 May;108(5):preceding 2001]

Gephyrin accumulates at specific plasmalemma loci during neuronal maturation in vitro

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