New instruments which facilitate rapid freezing at 83 K and 6 K

Escaig, J.

doi:10.1111/j.1365-2818.1982.tb00379.x

Cited by 217 publications

(149 citation statements)

References 14 publications

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“…Using a similar apparatus, Escaig measured, at the rear surface of a 40-#m tissue slice, a time of 1.5 ms to cool the sample from 293 to 253"K (19). The theoretical value for the same parameters, using the finite element method, is 0.25 ms (1).…”

Section: Discussionmentioning

confidence: 99%

“…nents) and its low time resolution for physiological events. Among the various cryotechniques (for review see reference 40), pressing living tissues against a copper block cooled by liquid helium to a temperature of ~ 15*K appears to be one of the quickest way of freezing thick biological materials for excellent morphological-preservation (1,12,17,19,43). Indeed, to capture rapid morphological changes that occur concomitantly with transient physiological processes, such as synaptic transmission, one might require cooling rates higher than those needed to prevent formation of visible ice crystals.…”

Section: Discussionmentioning

confidence: 99%

“…2-4 are micrographs from control muscles that illustrate the typical degree of preservation achieved in our experiments. The uppermost superficial layer of the muscle fibers (muscle--copper block interface), where the maximum heat transfer occurs, is well preserved (1,16,19,40). The depth of this layer, marked by the dotted line in Fig.…”

Section: Control Musclesmentioning

confidence: 99%

“…The quick-freezing device we used (Cryoblock mounted on a Cryofract 190, Reichert Jung S. A., Paris), previously described in detail, provides one of the quickest rates for freezing thick biological specimens with minimum precooling (19). With this device, slices of tissue 40-t~m thick can be frozen in 1.5 ms, and damage to the ultrastructure by ice crystals is not evident within the depth of 6-30 #m from the surface, depending on the specimen used.…”

Section: Methodsmentioning

confidence: 99%

“…We quick-froze nerve terminals using an improved quick-freezing apparatus (19), and we studied their morphology primarily on thin sections of cryosubstituted specimens, excised always from the medial edge of the muscle in order to keep constant the length of nerve so that their axons had similar conduction times. The ultrastructure was well preserved only in the outermost layer of tissue, 5-10 #m below the surface, that struck the copper block, and only terminals from this narrow region were examined.…”

mentioning

confidence: 99%

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

Torri-Tarelli¹,

Grohovaz²,

Fesce³

et al. 1985

The Journal of Cell Biology

151

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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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Section: Discussionmentioning

confidence: 99%