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. 1995 May;68(5):2156–2170. doi: 10.1016/S0006-3495(95)80398-X

Calcium transients in cerebellar granule cell presynaptic terminals.

W G Regehr 1, P P Atluri 1
PMCID: PMC1282121  PMID: 7612860

Abstract

Calcium ions act presynaptically to modulate synaptic strength and to trigger neurotransmitter release. Here we detect stimulus-evoked changes in residual free calcium ([Ca2+]i) in rat cerebellar granule cell presynaptic terminals. Granule cell axons, known as parallel fibers, and their associated boutons, were labeled with several calcium indicators. When parallel fibers were extracellularly activated with stimulus trains, calcium accumulated in the terminals, producing changes in the fluorescence of the indicators. During the stimulus train, the fluorescence change per pulse became progressively smaller with the high affinity indicators Fura-2 and calcium green-2 but remained constant with the low affinity dyes BTC and furaptra. In addition, fluorescence transients of high affinity dyes were slower than those of low affinity indicators, which appear to accurately report the time course of calcium transients. Simulations show that differences in the observed transients can be explained by the different affinities and off rates of the fluorophores. The return of [Ca2+]i to resting levels can be approximated by an exponential decay with a time constant of 150 ms. On the basis of the degree of saturation in the response of high affinity dyes observed during trains, we estimate that each action potential increases [Ca2+]i in the terminal by several hundred nanomolar. These findings indicate that in these terminals [Ca2+]i transients are much larger and faster than those observed in larger boutons, such as those at the neuromuscular junction. Such rapid [Ca2+]i dynamics may be found in many of the terminals in the mammalian brain that are similar in size to parallel fiber boutons.

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  1. Adler E. M., Augustine G. J., Duffy S. N., Charlton M. P. Alien intracellular calcium chelators attenuate neurotransmitter release at the squid giant synapse. J Neurosci. 1991 Jun;11(6):1496–1507. doi: 10.1523/JNEUROSCI.11-06-01496.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Almers W., Neher E. The Ca signal from fura-2 loaded mast cells depends strongly on the method of dye-loading. FEBS Lett. 1985 Nov 11;192(1):13–18. doi: 10.1016/0014-5793(85)80033-8. [DOI] [PubMed] [Google Scholar]
  3. Anderson K., Lai F. A., Liu Q. Y., Rousseau E., Erickson H. P., Meissner G. Structural and functional characterization of the purified cardiac ryanodine receptor-Ca2+ release channel complex. J Biol Chem. 1989 Jan 15;264(2):1329–1335. [PubMed] [Google Scholar]
  4. Augustine G. J., Adler E. M., Charlton M. P. The calcium signal for transmitter secretion from presynaptic nerve terminals. Ann N Y Acad Sci. 1991;635:365–381. doi: 10.1111/j.1749-6632.1991.tb36505.x. [DOI] [PubMed] [Google Scholar]
  5. Baylor S. M., Hollingworth S. Fura-2 calcium transients in frog skeletal muscle fibres. J Physiol. 1988 Sep;403:151–192. doi: 10.1113/jphysiol.1988.sp017244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berlin J. R., Konishi M. Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators. Biophys J. 1993 Oct;65(4):1632–1647. doi: 10.1016/S0006-3495(93)81211-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blatter L. A., Wier W. G. Intracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators indo-1 and fura-2. Biophys J. 1990 Dec;58(6):1491–1499. doi: 10.1016/S0006-3495(90)82494-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Blundon J. A., Wright S. N., Brodwick M. S., Bittner G. D. Residual free calcium is not responsible for facilitation of neurotransmitter release. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9388–9392. doi: 10.1073/pnas.90.20.9388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Delaney K. R., Tank D. W. A quantitative measurement of the dependence of short-term synaptic enhancement on presynaptic residual calcium. J Neurosci. 1994 Oct;14(10):5885–5902. doi: 10.1523/JNEUROSCI.14-10-05885.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Delaney K. R., Zucker R. S., Tank D. W. Calcium in motor nerve terminals associated with posttetanic potentiation. J Neurosci. 1989 Oct;9(10):3558–3567. doi: 10.1523/JNEUROSCI.09-10-03558.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fogelson A. L., Zucker R. S. Presynaptic calcium diffusion from various arrays of single channels. Implications for transmitter release and synaptic facilitation. Biophys J. 1985 Dec;48(6):1003–1017. doi: 10.1016/S0006-3495(85)83863-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fujita Y., Mynlieff M., Dirksen R. T., Kim M. S., Niidome T., Nakai J., Friedrich T., Iwabe N., Miyata T., Furuichi T. Primary structure and functional expression of the omega-conotoxin-sensitive N-type calcium channel from rabbit brain. Neuron. 1993 Apr;10(4):585–598. doi: 10.1016/0896-6273(93)90162-k. [DOI] [PubMed] [Google Scholar]
  13. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  14. Heidelberger R., Heinemann C., Neher E., Matthews G. Calcium dependence of the rate of exocytosis in a synaptic terminal. Nature. 1994 Oct 6;371(6497):513–515. doi: 10.1038/371513a0. [DOI] [PubMed] [Google Scholar]
  15. Hillyard D. R., Monje V. D., Mintz I. M., Bean B. P., Nadasdi L., Ramachandran J., Miljanich G., Azimi-Zoonooz A., McIntosh J. M., Cruz L. J. A new Conus peptide ligand for mammalian presynaptic Ca2+ channels. Neuron. 1992 Jul;9(1):69–77. doi: 10.1016/0896-6273(92)90221-x. [DOI] [PubMed] [Google Scholar]
  16. Hinkle P. M., Shanshala E. D., 2nd, Nelson E. J. Measurement of intracellular cadmium with fluorescent dyes. Further evidence for the role of calcium channels in cadmium uptake. J Biol Chem. 1992 Dec 15;267(35):25553–25559. [PubMed] [Google Scholar]
  17. Hollingworth S., Harkins A. B., Kurebayashi N., Konishi M., Baylor S. M. Excitation-contraction coupling in intact frog skeletal muscle fibers injected with mmolar concentrations of fura-2. Biophys J. 1992 Jul;63(1):224–234. doi: 10.1016/S0006-3495(92)81599-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Honoré T., Davies S. N., Drejer J., Fletcher E. J., Jacobsen P., Lodge D., Nielsen F. E. Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists. Science. 1988 Aug 5;241(4866):701–703. doi: 10.1126/science.2899909. [DOI] [PubMed] [Google Scholar]
  19. Iatridou H., Foukaraki E., Kuhn M. A., Marcus E. M., Haugland R. P., Katerinopoulos H. E. The development of a new family of intracellular calcium probes. Cell Calcium. 1994 Feb;15(2):190–198. doi: 10.1016/0143-4160(94)90058-2. [DOI] [PubMed] [Google Scholar]
  20. Kao J. P., Tsien R. Y. Ca2+ binding kinetics of fura-2 and azo-1 from temperature-jump relaxation measurements. Biophys J. 1988 Apr;53(4):635–639. doi: 10.1016/S0006-3495(88)83142-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Katz B., Miledi R. Tetrodotoxin-resistant electric activity in presynaptic terminals. J Physiol. 1969 Aug;203(2):459–487. doi: 10.1113/jphysiol.1969.sp008875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Klein M. G., Simon B. J., Szucs G., Schneider M. F. Simultaneous recording of calcium transients in skeletal muscle using high- and low-affinity calcium indicators. Biophys J. 1988 Jun;53(6):971–988. doi: 10.1016/S0006-3495(88)83178-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Konishi M., Berlin J. R. Ca transients in cardiac myocytes measured with a low affinity fluorescent indicator, furaptra. Biophys J. 1993 Apr;64(4):1331–1343. doi: 10.1016/S0006-3495(93)81494-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Konishi M., Hollingworth S., Harkins A. B., Baylor S. M. Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra. J Gen Physiol. 1991 Feb;97(2):271–301. doi: 10.1085/jgp.97.2.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Konishi M., Olson A., Hollingworth S., Baylor S. M. Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements. Biophys J. 1988 Dec;54(6):1089–1104. doi: 10.1016/S0006-3495(88)83045-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Llinás R., Sugimori M., Silver R. B. Microdomains of high calcium concentration in a presynaptic terminal. Science. 1992 May 1;256(5057):677–679. doi: 10.1126/science.1350109. [DOI] [PubMed] [Google Scholar]
  27. Mintz I. M., Adams M. E., Bean B. P. P-type calcium channels in rat central and peripheral neurons. Neuron. 1992 Jul;9(1):85–95. doi: 10.1016/0896-6273(92)90223-z. [DOI] [PubMed] [Google Scholar]
  28. Neher E., Augustine G. J. Calcium gradients and buffers in bovine chromaffin cells. J Physiol. 1992 May;450:273–301. doi: 10.1113/jphysiol.1992.sp019127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nowycky M. C., Pinter M. J. Time courses of calcium and calcium-bound buffers following calcium influx in a model cell. Biophys J. 1993 Jan;64(1):77–91. doi: 10.1016/S0006-3495(93)81342-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Palkovits M., Magyar P., Szentágothai J. Quantitative histological analysis of the cerebellar cortex in the cat. 3. Structural organization of the molecular layer. Brain Res. 1971 Nov;34(1):1–18. doi: 10.1016/0006-8993(71)90347-7. [DOI] [PubMed] [Google Scholar]
  31. Raju B., Murphy E., Levy L. A., Hall R. D., London R. E. A fluorescent indicator for measuring cytosolic free magnesium. Am J Physiol. 1989 Mar;256(3 Pt 1):C540–C548. doi: 10.1152/ajpcell.1989.256.3.C540. [DOI] [PubMed] [Google Scholar]
  32. Regehr W. G., Delaney K. R., Tank D. W. The role of presynaptic calcium in short-term enhancement at the hippocampal mossy fiber synapse. J Neurosci. 1994 Feb;14(2):523–537. doi: 10.1523/JNEUROSCI.14-02-00523.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Regehr W. G., Tank D. W. Calcium concentration dynamics produced by synaptic activation of CA1 hippocampal pyramidal cells. J Neurosci. 1992 Nov;12(11):4202–4223. doi: 10.1523/JNEUROSCI.12-11-04202.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Regehr W. G., Tank D. W. Selective fura-2 loading of presynaptic terminals and nerve cell processes by local perfusion in mammalian brain slice. J Neurosci Methods. 1991 Apr;37(2):111–119. doi: 10.1016/0165-0270(91)90121-f. [DOI] [PubMed] [Google Scholar]
  35. Regehr W. G., Tank D. W. The maintenance of LTP at hippocampal mossy fiber synapses is independent of sustained presynaptic calcium. Neuron. 1991 Sep;7(3):451–459. doi: 10.1016/0896-6273(91)90297-d. [DOI] [PubMed] [Google Scholar]
  36. Robitaille R., Adler E. M., Charlton M. P. Strategic location of calcium channels at transmitter release sites of frog neuromuscular synapses. Neuron. 1990 Dec;5(6):773–779. doi: 10.1016/0896-6273(90)90336-e. [DOI] [PubMed] [Google Scholar]
  37. Sala F., Hernández-Cruz A. Calcium diffusion modeling in a spherical neuron. Relevance of buffering properties. Biophys J. 1990 Feb;57(2):313–324. doi: 10.1016/S0006-3495(90)82533-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Simon S. M., Llinás R. R. Compartmentalization of the submembrane calcium activity during calcium influx and its significance in transmitter release. Biophys J. 1985 Sep;48(3):485–498. doi: 10.1016/S0006-3495(85)83804-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smith S. J., Buchanan J., Osses L. R., Charlton M. P., Augustine G. J. The spatial distribution of calcium signals in squid presynaptic terminals. J Physiol. 1993 Dec;472:573–593. doi: 10.1113/jphysiol.1993.sp019963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Swandulla D., Hans M., Zipser K., Augustine G. J. Role of residual calcium in synaptic depression and posttetanic potentiation: fast and slow calcium signaling in nerve terminals. Neuron. 1991 Dec;7(6):915–926. doi: 10.1016/0896-6273(91)90337-y. [DOI] [PubMed] [Google Scholar]
  41. Thastrup O., Cullen P. J., Drøbak B. K., Hanley M. R., Dawson A. P. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2466–2470. doi: 10.1073/pnas.87.7.2466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Williams M. E., Brust P. F., Feldman D. H., Patthi S., Simerson S., Maroufi A., McCue A. F., Veliçelebi G., Ellis S. B., Harpold M. M. Structure and functional expression of an omega-conotoxin-sensitive human N-type calcium channel. Science. 1992 Jul 17;257(5068):389–395. doi: 10.1126/science.1321501. [DOI] [PubMed] [Google Scholar]
  43. Winslow J. L., Duffy S. N., Charlton M. P. Homosynaptic facilitation of transmitter release in crayfish is not affected by mobile calcium chelators: implications for the residual ionized calcium hypothesis from electrophysiological and computational analyses. J Neurophysiol. 1994 Oct;72(4):1769–1793. doi: 10.1152/jn.1994.72.4.1769. [DOI] [PubMed] [Google Scholar]
  44. Wu L. G., Saggau P. Adenosine inhibits evoked synaptic transmission primarily by reducing presynaptic calcium influx in area CA1 of hippocampus. Neuron. 1994 May;12(5):1139–1148. doi: 10.1016/0896-6273(94)90321-2. [DOI] [PubMed] [Google Scholar]
  45. Wu L. G., Saggau P. Presynaptic calcium is increased during normal synaptic transmission and paired-pulse facilitation, but not in long-term potentiation in area CA1 of hippocampus. J Neurosci. 1994 Feb;14(2):645–654. doi: 10.1523/JNEUROSCI.14-02-00645.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yamada W. M., Zucker R. S. Time course of transmitter release calculated from simulations of a calcium diffusion model. Biophys J. 1992 Mar;61(3):671–682. doi: 10.1016/S0006-3495(92)81872-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

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