Abstract
We describe the design and operation of a machine that freezes biological tissues by contact with a cold metal block, which incorporates a timing circuit that stimulates frog neuromuscular junctions in the last few milliseconds before thay are frozen. We show freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synpatic vesicles caught in the act of exocytosis. We use 4-aminopyridine (4-AP) to increase the number of transmitter quanta discharged with each nerve impulse, and show that the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that one vesicle undergoes exocytosis for each quantum that is discharged. We perform statistical analyses on the spatial distribution of synaptic vesicle discharge sites along the "active zones" that mark the secretory regions of these nerves, and show that individual vesicles fuse with the plasma membrane independent of one another, as expected from physiological demonstrations that quanta are discharged independently. Thus, the utility of quick- freezing as a technique to capture biological processes as evanescent as synaptic transmission has been established. An appendix describes a new capacitance method to measure freezing rates, which shows that the "temporal resolution" of our quick-freezing technique is 2 ms or better.
Full Text
The Full Text of this article is available as a PDF (4.4 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- BIRKS R., HUXLEY H. E., KATZ B. The fine structure of the neuromuscular junction of the frog. J Physiol. 1960 Jan;150:134–144. doi: 10.1113/jphysiol.1960.sp006378. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bevan S. Sub-miniature end-plate potentials at untreated frog neuromuscular junctions. J Physiol. 1976 Jun;258(1):145–155. doi: 10.1113/jphysiol.1976.sp011411. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Branisteanu D. D., Miyamoto M. D., Volle R. L. Effects of physiologic alterations on binomial transmitter release at magnesium-depressed neuromuscular junctions. J Physiol. 1976 Jan;254(1):19–37. doi: 10.1113/jphysiol.1976.sp011218. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ceccarelli B., Hurlbut W. P., Mauro A. Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J Cell Biol. 1973 May;57(2):499–524. doi: 10.1083/jcb.57.2.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DEL CASTILLO J., ENGBAEK L. The nature of the neuromuscular block produced by magnesium. J Physiol. 1954 May 28;124(2):370–384. doi: 10.1113/jphysiol.1954.sp005114. [DOI] [PMC free article] [PubMed] [Google Scholar]
- FATT P., KATZ B. Spontaneous subthreshold activity at motor nerve endings. J Physiol. 1952 May;117(1):109–128. [PMC free article] [PubMed] [Google Scholar]
- Heuser J. E., Reese T. S., Landis D. M. Functional changes in frog neuromuscular junctions studied with freeze-fracture. J Neurocytol. 1974 Mar;3(1):109–131. doi: 10.1007/BF01111936. [DOI] [PubMed] [Google Scholar]
- Heuser J. E., Reese T. S., Landis D. M. Preservation of synaptic structure by rapid freezing. Cold Spring Harb Symp Quant Biol. 1976;40:17–24. doi: 10.1101/sqb.1976.040.01.004. [DOI] [PubMed] [Google Scholar]
- JENKINSON D. H. The antagonism between tubocurarine and substances which depolarize the motor end-plate. J Physiol. 1960 Jul;152:309–324. doi: 10.1113/jphysiol.1960.sp006489. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jan Y. N., Jan L. Y., Dennis M. J. Two mutations of synaptic transmission in Drosophila. Proc R Soc Lond B Biol Sci. 1977 Jul 28;198(1130):87–108. doi: 10.1098/rspb.1977.0087. [DOI] [PubMed] [Google Scholar]
- KATZ B., MILEDI R. THE MEASUREMENT OF SYNAPTIC DELAY, AND THE TIME COURSE OF ACETYLCHOLINE RELEASE AT THE NEUROMUSCULAR JUNCTION. Proc R Soc Lond B Biol Sci. 1965 Feb 16;161:483–495. doi: 10.1098/rspb.1965.0016. [DOI] [PubMed] [Google Scholar]
- Letinsky M. S., Fischbeck K. H., McMahan U. J. Precision of reinnervation of original postsynaptic sites in frog muscle after a nerve crush. J Neurocytol. 1976 Dec;5(6):691–718. doi: 10.1007/BF01181582. [DOI] [PubMed] [Google Scholar]
- Marty A., Neild T., Ascher P. Voltage sensitivity of acetylcholine currents in Aplysia neurones in the presence of curare. Nature. 1976 Jun 10;261(5560):501–503. doi: 10.1038/261501a0. [DOI] [PubMed] [Google Scholar]
- Nastuk W. L., Liu J. H. Muscle postjunctional membrane: changes in chemosensitivity produced by calcium. Science. 1966 Oct 14;154(3746):266–267. doi: 10.1126/science.154.3746.266. [DOI] [PubMed] [Google Scholar]
- Pfenninger K., Akert K., Moor H., Sandri C. Freeze-fracturing of presynaptic membranes in the central nervous system. Philos Trans R Soc Lond B Biol Sci. 1971 Jun 17;261(839):387–387. doi: 10.1098/rstb.1971.0071. [DOI] [PubMed] [Google Scholar]
- Pfenninger K., Akert K., Moor H., Sandri C. The fine structure of freeze-fractured presynaptic membranes. J Neurocytol. 1972 Sep;1(2):129–149. doi: 10.1007/BF01099180. [DOI] [PubMed] [Google Scholar]
- Wernig A., Stirner H. Quantum amplitude distributions point to functional unity of the synaptic 'active zone'. Nature. 1977 Oct 27;269(5631):820–822. doi: 10.1038/269820a0. [DOI] [PubMed] [Google Scholar]
- Whittaker V. P., Essman W. B., Dowe G. H. The isolation of pure cholinergic synaptic vesicles from the electric organs of elasmobranch fish of the family Torpedinidae. Biochem J. 1972 Jul;128(4):833–845. doi: 10.1042/bj1280833. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yeh J. Z., Oxford G. S., Wu C. H., Narahashi T. Dynamics of aminopyridine block of potassium channels in squid axon membrane. J Gen Physiol. 1976 Nov;68(5):519–535. doi: 10.1085/jgp.68.5.519. [DOI] [PMC free article] [PubMed] [Google Scholar]