Summary
A quantitative study was made of the effects of 24 h continuous stimulation on the morphology of the frog neuromuscular junction. The synaptic vesicle concentration in the nerve endings of frog sartorius muscles stimulatedin vitro for 24 h at 2 Hz was the same as that in controls stimulated for only 0.3 h at 2 Hz. The control preparations were either freshly dissected or maintained at restin vitro for 23 h prior to stimulation. Chronically stimulated terminals differed from their controls only in having more cisternae and fewer dense cored vesicles. Varying the lengths of the nerves to both chronically stimulated andin vitro control muscles had little effect on the morphology of the nerve endings.
Continuous recording of muscle twitch tension demonstrated that neurotransmission was effective throughout the 24 h period of stimulation. Additional evidence that nerve failure or degeneration was not a factor in the results came from a second set of control and chronically stimulated preparations that were tetanized at 30 Hz for 0.3 h before fixation. Changes attributable to rapid stimulation were evident in 87 to 100% of their nerve terminals.
Although the distribution of membrane among various membrane organelles differed from one treatment group to another, the total amount of measurable membrane in the nerve terminals was the same in all of the treatment groups; that is, the total amount of membrane was not altered by maintenancein vitro, chronic stimulation at 2 Hz, rapid stimulation at 30 Hz, reduced nerve length, or any tested combination of these treatments. This conservation of total membrane suggests that membrane exchange between axon and nerve terminal occurs at a relatively slow rate which is unaffected by synaptic activity, and that the local mechanism for recycling synaptic vesicle membrane in frog neuromuscular junctions is more autonomous and durable than has been suspected.
Similar content being viewed by others
References
Birks, R. I., Huxley, H. E. &Katz, B. (1960a) The fine structure of the neuromuscular junction of the frog.Journal of Physiology 150, 134–44.
Birks, R. I., Katz, B. &Miledi, R. (1960b) Physiological and structural changes at the amphibian neuromuscular junction, in the course of nerve degeneration.Journal of Physiology 150, 145–68.
Blaustein, M. P., Ratzlaff, R. W., Kendrick, N. C. &Schweitzer, E. S. (1978) Calcium buffering in presynaptic nerve terminals. I. Evidence for a nonmitochondrial ATP-dependent sequestration mechanism.Journal of General Physiology 72, 15–41.
Boyne, A. F., Bohan, T. P. &Williams, T. H. (1975) Changes in cholinergic synaptic vesicle populations and the ultra structure of the nerve terminal membranes ofNarcine brasiliensis electric organ stimulated to fatiguein vivo.Journal of Cell Biology 67, 814–25.
Ceccarelli, B., Hurlbut, W. P. &Mauro, A. (1972) Depletion of vesicles from frog neuromuscular junction by prolonged tetanic stimulation.Journal of Cell Biology 54, 30–8.
Ceccarelli, B., Hurlbut, W. P. &Mauro, A. (1973) Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction.Journal of Cell Biology 57, 499–524.
Cochran, W. G. (1954) The combination of estimates from different experiments.Biometrics 10, 101–29.
Conover, W. J. (1971)Practical Nonparametric Statistics, pp. 309–14. New York: John Wiley and Sons, Inc.
Dahlstrom, A. (1967) The transport of noradrenaline between two simultaneously performed ligations of the sciatic nerves of rat and cat.Acta physiologica scandinavica 69, 158–66.
Del Castillo, J. &Katz, B. (1954a) Quantal components of the endplate potential.Journal of Physiology 124, 560–73.
Del Castillo, J. &Katz, B. (1954b) Statistical factors involved in neuromuscular facilitation and depression.Journal of Physiology 124, 574–85.
Droz, B., Rambourg, A. &Koenig, H. L. (1975) The smooth endoplasmic reticulum: Structure and role in the renewal of axonal membrane and synaptic vesicles by fast axonal transport.Brain Research 93, 1–13.
Edstrom, A. &Hanson, M. (1973) Temperature effects on fast axoplasmic transport of proteinsin vitro in frog sciatic nerves.Brain Research 58, 345–54.
Fatt, P. &Katz, B. (1951) An analysis of the end-plate potential recorded with an intra-cellular electrode.Journal of Physiology 115, 320–70.
Fatt, P. &Katz, B. (1952) Spontaneous subthreshold activity at motor nerve endings.Journal of Physiology 117, 109–28.
Fried, R. C. &Blaustein, M. P. (1978) Retrieval and recycling of synaptic vesicle membrane in pinched-off nerve terminals (synaptosomes).Journal of Cell Biology 78, 685–700.
Gennaro, J. R., Jr, Nastuk, W. L. &Rutherford, D. T. (1978) Reversible depletion of synaptic vesicles induced by application of high external potassium to the frog neuromuscular junction.Journal of Physiology 280, 237–47.
Grafstein, B. &Forman, D. S. (1980) Intracellular transport in neurons.Physiological Reviews 60, 1167–283.
Hackenbrock, C. R. &Caplan, A. I. (1969) Ion-induced ultrastructural transformations in isolated mitochondria.Journal of Cell Biology 42, 221–34.
Harris, A. J. &Miledi, R. (1972) A study of frog muscle maintained in organ culture.Journal of Physiology 221, 207–26.
Henkart, M. P., Reese, T. S. &Brinley, F. J., Jr (1978) Endoplasmic reticulum sequesters calcium in squid giant axon.Science 202, 1300–3.
Heuser, J. E. (1976) Morphology of synaptic vesicle discharge and reformation at the frog neuromuscular junction. InMotor Innervation of Muscle (edited byThesleff, S.), pp. 51–115. New York: Academic Press.
Heuser, J. E. &Miledi, R. (1971) Effect of lanthanum ions on function and structure of frog neuromuscular junctions.Proceedings of the Royal Society of London, Series B 179, 247–60.
Heuser, J. E. &Reese, T. S. (1973) Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction.Journal of Cell Biology 57, 315–44.
Heuser, J. E., Katz, B. &Miledi, R. (1971) Structural and functional changes of frog neuromuscular junctions in high calcium solution.Proceedings of the Royal Society of London, Series B 178, 407–15.
Holtzman, E. (1976)Lysosomes: A Survey. Wein: Springer-Verlag.
Holtzman, E. (1977) The origin and fate of secretory packages, especially synaptic vesicles.Neuroscience 2, 327–55.
Holtzman, E. &Mercurio, A. M. (1980) Membrane circulation in neurons and photo-receptors: some unresolved issues.International Review of Cytology 67, 1–67.
Holtzman, E., Freeman, A. R. &Kashner, L. A. (1971) Stimulation-dependent alterations in peroxidase uptake at lobster neuromuscular junctions.Science 173, 733–6.
Hunt, C. C. &Nelson, P. G. (1965) Structural and functional changes in the frog sympathetic ganglion following cutting of the presynaptic nerve fibres.Journal of Physiology 177, 1–20.
Jankowska, E., Lubinska, L. &Niemierko, S. (1969) Translocation of AChE-containing particles in the axoplasm during nerve activity.Comparative Biochemistry and Physiology 28, 907–13.
Karnovsky, M. J. (1971) In11th Annual Meeting of American Society of Cell Biology, Abstract 284, p. 146.
Katz, B. &Kuffler, S. W. (1941) Multiple motor innervation of the frog's sartorius muscle.Journal of Neurophysiology 4, 209–23.
Katz, B. &Miledi, R. (1967) A study of synaptic transmission in the absence of nerve impulses.Journal of Physiology 192, 407–36.
Korneliussen, H. (1972a) Ultrastructure of normal and stimulated motor endplates: with comments on the origin and fate of synaptic vesicles.Zeitschrift fur Zellforschung und mikroskopische Anatomie 130, 28–57.
Korneliussen, H. (1972b) Elongated profiles of synaptic vesicles in motor endplates: effects of fixative variations.Journal of Neurocytology 1, 279–96.
Kristensson, K., Olsson, Y. &Sjostrand, J. (1971) Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve.Brain Research 32, 399–406.
Krnjevic, K. &Miledi, R. (1959) Presynaptic failure of neuromuscular propagation in rats.Journal of Physiology 149, 1–22.
LaVail, J. H. &LaVail, M. M. (1974) The retrograde intra-axonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopic study.Journal of Comparative Neurology 157, 303–58.
LaVail, J. H., Rapisardi, S. &Sugino, I. K. (1980) Evidence against the smooth endoplasmic reticulum as a continuous channel for the retrograde axonal transport of horseradish peroxidase.Brain Research 191, 3–20.
Llinas, R. &Nicholson, C. (1975) Calcium role in depolarization-secretion coupling: an aequorin study in squid giant synapses.Proceedings of the National Academy of Sciences 72, 187–90.
Lubinska, L. &Niemierko, S. (1971) Velocity and intensity of bidirectional migration of acetylcholinesterase in transected nerves.Brain Research 27, 329–42.
Lux, H. D., Schubert, P., Kreutzberg, G. W. &Globus, A. (1970) Excitation and axonal flow: autoradiographic study on motorneurons intracellularly injected with a3H-amino acid.Experimental Brain Research 10, 197–204.
Lynch, K. (1980) Stimulation-induced reduction of large dense core vesicle numbers in cholinergic motor nerve endings.Brain Research 194, 249–54.
Martin, A. R. (1955) A further study of the statistical composition of the end plate potential.Journal of Physiology 130, 114–22.
Miledi, R. &Potter, L. T. (1971) Acetylcholine receptors in muscle fibres.Nature 233, 599–604.
Miller, R. J., Jr. (1966)Simultaneous Statistical Inference, pp. 67–70. New York: McGraw Hill.
Model, P. G., Highstein, S. M. &Bennett, M. V. L. (1975) Depletion of vesicles and fatigue of transmission at a vertebrate central synapse.Brain Research 98, 209–28.
Peachey, L. (1964) Electron microscopic observations on the accumulation of divalent cations in intramitochondrial granules.Journal of Cell Biology 20, 95–109.
Pysh, J. J. &Wiley, R. G. (1974) Synaptic vesicle depletion and recovery in cat sympathetic ganglia electrically stimulatedin vivo: evidence for transmitter secretion by exocytosis.Journal of Cell Biology 60, 365–74.
Ralston, H. J. &Libet, B. (1953) Effect of stretch on action potentials of voluntary muscle.American Journal of Physiology 173, 449–55.
Reinecke, M. &Walther, C. (1978) Aspects of turnover and biogenesis of synaptic vesicles at locust neuromuscular junctions as revealed by zinc iodide-osmium tetroxide (ZIO) reacting with intravesicular SH-groups.Journal of Cell Biology 78, 839–55.
Schacher, S. M., Holtzman, E. &Hood, D. C. (1976) Synaptic activity of frog retinal photoreceptors: a peroxidase uptake study.Journal of Cell Biology 70, 178–92.
Schaeffer, S. F. &Raviola, E. (1978) Membrane recycling in the cone cell endings of the turtle retina.Journal of Cell Biology 79, 802–25.
Stelzner, D. J. (1971) The relationship between synaptic vesicles, Golgi apparatus and smooth endoplasmic reticulum: a developmental study using the zinc iodide-osmium technique.Zeitschrift für Zellforschung und mikroskopische Anatomie 120, 332–45.
Teichberg, S., Holtzman, E., Crain, S. M. &Peterson, E. R. (1975) Circulation and turnover of synaptic membrane in cultured spinal cord neurons.Journal of Cell Biology 67, 215–30.
Tsukita, S. &Ishikawa, H. (1980) The movements of membranous organelles in axons. Electron microscopic identification of anterogradely and retrogradely transported organelles.Journal of Cell Biology 84, 513–30.
Zimmermann, H. &Denston, C. R. (1977) Recycling of synaptic vesicles in the cholinergic synapses of theTorpedo electric organ during induced transmitter release.Neuroscience 2, 695–714.
Zimmermann, H. &Whittaker, V. P. (1974) Different recovery rates of electrophysiological, biochemical and morphological parameters in cholinergic synapses ofTorpedo electric organ after stimulation.Journal of Neurochemistry 22, 1109–14.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lynch, K. The effects of chronic stimulation on the morphology of the frog neuromuscular junction. J Neurocytol 11, 81–107 (1982). https://doi.org/10.1007/BF01258006
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01258006