Functional changes in frog neuromuscular junctions studied with freeze-fracture

Heuser, John E.; Reese, Thomas S.; Landis, Dennis M.D.

doi:10.1007/bf01111936

Cited by 501 publications

(306 citation statements)

References 20 publications

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“…Nerve terminals imaged by freeze fractu re exhibited large transmembrane particles at release sites (Pumplin et al, 1981;Heuser et al, 1974Heuser et al, , 1979Ceccarelli et al, 1979), which were believed to reflect the transmembrane regions of calcium channels. However, many other putative membrane proteins, including syntaxin (Bennett et al, 1992;O'Connor et al, 1993;Horikawa et al, 1993), neurexin (Petrenko et al, 1991;Petrenko, 1993), and calcium-activated potassium channels (Roberts et al, 1990;Robitaille et al, 1993), are also localized at or near the transmitter release site, and these may also generate particles in the freeze-fracture replica.…”

Section: Analysis Of Calcium Channel Distributionmentioning

confidence: 99%

Localization of individual calcium channels at the release face of a presynaptic nerve terminal

Haydon

Henderson

Stanley

1994

Neuron

116

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SummaryStudies using biophysical techniques suggest a highly structured organization of calcium channels at the presynaptic transmitter release face (LEnds et al., 1981; Stanley, 1993), but it has not as yet proved possible to localize identified channels at the required nanometer level of resolution. We have used atomic force microscopy on the calyx-type nerve terminal of the chick ciliary ganglion to localize single calcium channels tagged via biotinylated ~o-conotoxin GVIA to avidin-coated 30 nm gold particles. Calcium channels were in low (modal value approximately ~ 1 per p,m ~) and high (modal value = 55 per l~m 2) density areas and exhibited a prominent interchannel spacing of 40 nm, indicating an intermolecular linkage. Particles were observed in clusters and short linear or parallel linear arrays, groupings that may reflect calcium channel organization at the transmitter release site.

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Section: Analysis Of Calcium Channel Distributionmentioning

confidence: 99%

Localization of individual calcium channels at the release face of a presynaptic nerve terminal

Haydon

Henderson

Stanley

1994

Neuron

116

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“…We simulated a single AZ, which consists of a transverse valley extending across the width of the terminal with Ca 2ϩ channels situated on the walls bordered by two rows of synaptic vesicles (Heuser et al, 1974;Harlow et al, 2001). We have estimated the shape and geometrical parameters of the AZ based on a number of morphological studies (Pawson et al, 1998;Harlow et al, 2001;Stanley et al, 2003) (see Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Several putative Ca 2ϩ channels are located within 100 nm of each vesicle (Heuser et al, 1974;Pumplin et al, 1981), so a high degree of channel cooperativity is possible. However, indirect electrophysiological analysis (Yoshikami et al, 1989;Augustine et al, 1991) and a recent Ca 2ϩ imaging study (Wachman et al, 2004) are consistent with only a few channels opening with each action potential (AP), suggesting low cooperativity.…”

Section: Introductionmentioning

confidence: 99%

Ca²⁺from One or Two Channels Controls Fusion of a Single Vesicle at the Frog Neuromuscular Junction

Shahrezaei¹,

Cao²,

Delaney³

2006

J. Neurosci.

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“…Whatever the cause of the difference between the two sets of results, it appears that in those regions of the muscles where an adequate degree of tissue preservation is achieved the plane of fracture is quite different from the one normally obtained in chemically fixed and cryoprotected specimens (6,7,27). This is not surprising since there are no convincing a priori reasons to believe that in a quasi-vitrified portion of a cell the inner membrane domain should be more prone to be freeze-cleaved (22).…”

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confidence: 99%

Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

Torri-Tarelli¹,

Grohovaz²,

Fesce³

et al. 1985

The Journal of Cell Biology

149

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We applied the quick-freezing technique to investigate the precise temporal coincidence between the onset of quantal secretion and the appearance of fusions of synaptic vesicles with the prejunctional membrane. Frog cutaneous pectoris nerve-muscle preparations were soaked in modified Ringer's solution with 1 mM 4-aminopyridine, 10 mM Ca 2+, and 10 -4 M d-Tubocurarine and quick-frozen 1-10 ms after a single supramaximal shock. The frozen muscles were then either freeze-fractured or cryosubstituted in acetone with 13% OsO4 and processed for thin section electron microscopy. Temporal resolution of <1 ms can be achieved using a quick-freeze device that increases the rate of freezing of the muscle after it strikes the chilled copper block (15°K) and that minimizes the precooling of the muscle during its descent toward the block. We minimized variations in transmission time by examining thin sections taken only from the medial edge of the muscle, which was at a fixed distance from the point of stimulation of the nerve. The ultrastructure of the cryosubstituted preparations was well preserved to a depth of 5-10/~m, and within this narrow band vesicles were found fused with the axolemma after a minimum delay of 2.5 ms after stimulation of the nerve. Since the total transmission time to this edge of the muscle was ~3 ms, these results indicate that the vesicles fuse with the axolemma precisely at the same time the quanta are released.Freeze-fracture does not seem to be an adequate experimental technique for this work because in the well-preserved band of the muscle the fracture plane crosses, but does not cleave, the inner hydrophobic domain of the plasmalemma. Fracture faces may form in deeper regions of the muscle where tissue preservation is unsatisfactory and freezing is delayed.Neurotransmitters are released from nerve terminals in two ways: by the continuous leakage of individual molecules across the nerve terminal membrane (23,33,41,50) and by synchronous secretion of few thousands molecules in packages, or quanta (14,15,20). Quantal secretion causes the changes in membrane potential that excite the postsynaptic cell, and transmission at a synapse is critically dependent upon this mechanism of release. The vesicle hypothesis holds that each quantum is confined within one of the synaptic vesicles that populate the nerve terminal and is released by exocytosis when the membrane of that vesicle fuses with the axolemma at one of the many specialized regions called "active zones" (8,13,51).A number of workers have shown that at frog neuromuscular junctions vesicles fuse with the axolemma of stimulated 1386 terminals and some of the remaining vesicles become labeled with extracellular tracers (9)(10)(11)25). Although these observations indicate that vesicles fuse with and are recovered from the axolemma when quanta are actively secreted, the slowness of chemical fixation precludes demonstrating that vesicle fusion and transmitter release are coincident. The images of fusion seen after chemical fixation represent...

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Functional changes in frog neuromuscular junctions studied with freeze-fracture

Cited by 501 publications

References 20 publications

Localization of individual calcium channels at the release face of a presynaptic nerve terminal

Localization of individual calcium channels at the release face of a presynaptic nerve terminal

Ca²⁺from One or Two Channels Controls Fusion of a Single Vesicle at the Frog Neuromuscular Junction

Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

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Functional changes in frog neuromuscular junctions studied with freeze-fracture

Cited by 501 publications

References 20 publications

Localization of individual calcium channels at the release face of a presynaptic nerve terminal

Localization of individual calcium channels at the release face of a presynaptic nerve terminal

Ca2+from One or Two Channels Controls Fusion of a Single Vesicle at the Frog Neuromuscular Junction

Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

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Ca²⁺from One or Two Channels Controls Fusion of a Single Vesicle at the Frog Neuromuscular Junction