Kuwait Medical Electronic Library

Differential Release of Exocytosis Marker Dyes Indicates Stimulation-Dependent Regulation of Synaptic Activity

Andreas W Henkel 1, Abdeslam Mouihate 1, Oliver Welzel 2

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02 October 2019

doi: 10.3389/fnins.2019.01047

Abstract

There is a general consensus that synaptic vesicular release by a full collapse process is the primary machinery of synaptic transmission. However, competing view suggests that synaptic vesicular release operates via a kiss-and-run mechanism. By monitoring the release dynamics of a synaptic vesicular marker, FM1-43 from individual synapses in hippocampal neurons, we found evidence that the release of synaptic vesicle was delayed by several seconds after the start of field stimulation. This phenomenon was associated with modified opening kinetics of fusion pores. Detailed analysis revealed that some synapses were completely inactive for a few seconds after stimulation, despite immediate calcium influx. This delay in vesicular release was modulated by various stimulation protocols and different frequencies, indicating an activity-dependent regulation mechanism for neurotransmitter exocytosis. Staurosporine, a drug known to induce "kiss-and-run" exocytosis, increased the proportion of delayed synapses as well as the delay duration, while fluoxetine acted contrarily. Besides being a serotonin reuptake inhibitor, it directly enhanced vesicle mobilization and reduced synaptic fatigue. Exocytosis was never delayed, when it was monitored with pH-sensitive probes, synaptopHlourin and αSyt-CypHerE5 antibody, indicating an instantaneous formation of a fusion pore that allowed rapid equilibration of vesicular lumenal pH but prevented FM1-43 release because of its slow dissociation from the inner vesicular membrane. Our observations suggest that synapses operate via a sequential "kiss-and-run" and "full-collapse" exocytosis mechanism. The initially narrow vesicular pore allows the equilibration of intravesicular pH which then progresses toward full fusion, causing FM1-43 release.

Keywords: FM1-43; exocytosis; fusion pore; hippocampal neurons; kiss-and-run.

Figures

FIGURE 1 (A) Three examples of synaptic classes, selected by SynoExa according to specified kinetic courses and fitting functions were overlaid onto mean traces during the stimulation period. Left panel: “Classical synapses” derived from n = 453 individual synapses, r = 0.997; middle panel: “Two-speed synapses,” n = 342, tau1: r = 0.998, tau2: r = 0.999; and right panel: “Linear synapses,” n = 126, r = 0.999. (B) Pseudo-colored image series (imaging rate: 1 Hz) shows a single delayed synapse that started destaining 3 s after start of stimulation (red bar). (C) Hippocampal synapses, stained with FM1-43. Individual spot intensities were detected and extracted with ImageStar (red box markers), and stored in a database together with their x–y positions. (D) Fluorescent traces, derived from individual spots, normalized to start of stimulation, overlaid onto the mean trace (black–red–black). Left panel, Traces from all identified spots; right panel, selected traces from synapses (VAS) with >0.35 dF/Fmax FM1-43 release.

FIGURE 2 (A) Fluorescent spots from Figure 1C after background subtraction. The traces from two labeled synapses are shown in the inset. Inset: classical synapse (1) and a delayed onset synapse (2). (B) Histogram of first-order derivatives of mean traces indicate Dt distribution of presented kinetic subpopulations as indicated by color. (C) Four panels showing individual synaptic fluorescence traces with increasing Dt and corresponding single exponential fits to trace sections after the start of FM1-43 release. Pre-stimulation sections were fitted with linear regression lines. (D) Comparison of two neighboring synapses with different Dt. The graph shows overlaid traces from regions, indicated in the images.

FIGURE 3 (A) Quantification of specified kinetic synapse classes in two subsequent staining/destaining cycles and effect of fluoxetine on class change in between both stimulation cycles (9 control experiments, 10,569 synapses; 11 fluoxetine experiments, and 11,299 synapses). ANOVA analysis indicated significant (p < 0.001) differences between the three groups in all five experimental setups. (B) Green color indicates staining of synapses with FM1-43, while red color shows direct subsequent stimulation-dependent destaining. Two single synapses (arrows) shows no destaining during the 1st stimulation cycle but recovered, and destained in the 2nd cycle. (C) Correlation plot of FM1-43 release between the 1st and the 2nd staining/destaining cycles (n = 424 matched synapses). (D) Direct comparison of individual synapses in subsequent staining/destaining cycles. Upper traces show the course of fluorescence and lower traces their corresponding first-order derivatives. First cycle (red), 2nd cycle (cyan), and stimulation period (red bar). The image pairs and the traces were directly obtained with SynCom Ver. 1.61, software. (E,F) The pie-charts display the differences between two successive staining/destaining cycles when no drug (control) or 10 μM fluoxetine was administered between the cycles, control n = 424, fluoxetine n = 387 synapses. (E) Differences in FM1-43 dye release from synapses between 1st and 2nd stimulation cycle. (F) Variances in the percentage of synapses with delayed release onset (DelSys) between 1st and 2nd stimulation cycle.

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KMEL References

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References

1. Adie E. J., Kalinka S., Smith L., Francis M. J., Marenghi A., Cooper M. E., et al. (2002). A pH-sensitive fluor, CypHer 5, used to monitor agonist-induced G protein-coupled receptor internalization in live cells. Biotechniques 33 1156–1157. - PubMed
1. Albillos A., Dernick G., Horstmann H., Almers W., Alvarez de Toledo G., Lindau M. (1997). The exocytotic event in chromaffin cells revealed by patch amperometry. Nature 389 509–512. 10.1038/39081 - DOI - PubMed
1. Balseiro-Gomez S., Flores J. A., Acosta J., Ramirez-Ponce M. P., Ales E. (2016). Transient fusion ensures granule replenishment to enable repeated release after IgE-mediated mast cell degranulation. J. Cell Sci. 129 3989–4000. - PubMed
1. Becherer U., Guatimosim C., Betz W. (2001). Effects of staurosporine on exocytosis and endocytosis at frog motor nerve terminals. J. Neurosci. 21 782–787. 10.1523/jneurosci.21-03-00782.2001 - DOI - PMC - PubMed
1. Betz W. J., Bewick G. S. (1992). Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science 255 200–203. 10.1126/science.1553547 - DOI - PubMed
1. Betz W. J., Mao F., Bewick G. S. (1992). Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. J. Neurosci. 12 363–375. 10.1523/jneurosci.12-02-00363.1992 - DOI - PMC - PubMed
1. Blank P. S., Vogel S. S., Malley J. D., Zimmerberg J. (2001). A kinetic analysis of calcium-triggered exocytosis. J. Gen. Physiol. 118 145–156. 10.1085/jgp.118.2.145 - DOI - PMC - PubMed
1. Castillo P. E. (2012). Presynaptic LTP and LTD of excitatory and inhibitory synapses. Cold Spring Harb Persp. Biol. 4:a005728. 10.1101/cshperspect.a005728 - DOI - PMC - PubMed
1. Ceccarelli B., Hurlbut W. P. (1980). Vesicle hypothesis of the release of quanta of acetylcholine. Physiol. Rev. 60 396–441. 10.1152/physrev.1980.60.2.396 - DOI - PubMed
1. Chang C. W., Chiang C. W., Jackson M. B. (2017). Fusion pores and their control of neurotransmitter and hormone release. J. Gen. Physiol. 149 301–322. 10.1085/jgp.201611724 - DOI - PMC - PubMed
1. Cohen J. E. (1988). Estimating the effects of successful malarial control programmes on mortality. Popul. Bull. UN 6–26. - PubMed
1. Elhamdani A., Azizi F., Artalejo C. R. (2006). Double patch clamp reveals that transient fusion (kiss-and-run) is a major mechanism of secretion in calf adrenal chromaffin cells: high calcium shifts the mechanism from kiss-and-run to complete fusion. J. Neurosci. 26 3030–3036. 10.1523/jneurosci.5275-05.2006 - DOI - PMC - PubMed
1. Emes R. D., Grant S. G. (2012). Evolution of synapse complexity and diversity. Annu. Rev. Neurosci. 35 111–131. 10.1146/annurev-neuro-062111-150433 - DOI - PubMed
1. Fernandes D., Carvalho A. L. (2016). Mechanisms of homeostatic plasticity in the excitatory synapse. J. Neurochem. 139 973–996. 10.1111/jnc.13687 - DOI - PubMed
1. Fisher R. J., Pevsner J., Burgoyne R. D. (2001). Control of fusion pore dynamics during exocytosis by Munc18. Science 291 875–878. 10.1126/science.291.5505.875 - DOI - PubMed
1. Gan Q., Watanabe S. (2018). Synaptic vesicle endocytosis in different model systems. Front. Cell Neurosci. 12:171. 10.3389/fncel.2018.00171 - DOI - PMC - PubMed
1. Granseth B., Odermatt B., Royle S. J., Lagnado L. (2009). Comment on "The dynamic control of kiss-and-run and vesicular reuse probed with single nanoparticles. Science 325:1499. 10.1126/science.1175790 - DOI - PubMed
1. Gu Z., Lamb P. W., Yakel J. L. (2012). Cholinergic coordination of presynaptic and postsynaptic activity induces timing-dependent hippocampal synaptic plasticity. J. Neurosci. 32 12337–12348. 10.1523/JNEUROSCI.2129-12.2012 - DOI - PMC - PubMed
1. Harata N. C., Choi S., Pyle J. L., Aravanis A. M., Tsien R. W. (2006). Frequency-dependent kinetics and prevalence of kiss-and-run and reuse at hippocampal synapses studied with novel quenching methods. Neuron 49 243–256. 10.1016/j.neuron.2005.12.018 - DOI - PubMed
1. He L., Wu X. S., Mohan R., Wu L. G. (2006). Two modes of fusion pore opening revealed by cell-attached recordings at a synapse. Nature 444 102–105. 10.1038/nature05250 - DOI - PubMed
1. Henkel A. W. (2015). Analysis of kinetically distinguished synaptic subtypes in hippocampal neurons. Biophys. J. 108 101a–101a.
1. Henkel A. W., Alali H., Devassy A., Alawadi M. M., Redzic Z. B. (2014). Antagonistic interactions between dexamethasone and fluoxetine modulate morphodynamics and expression of cytokines in astrocytes. Neuroscience 280 318–327. 10.1016/j.neuroscience.2014.09.012 - DOI - PubMed
1. Henkel A. W., Betz W. J. (1995). Staurosporine blocks evoked release of FM1-43 but not acetylcholine from frog motor nerve terminals. J. Neurosci. 15 8246–8258. 10.1523/jneurosci.15-12-08246.1995 - DOI - PMC - PubMed
1. Henkel A. W., Kang G., Kornhuber J. (2001). A common molecular machinery for exocytosis and the ‘kiss-and-run’ mechanism in chromaffin cells is controlled by phosphorylation. J. Cell Sci. 114 4613–4620. - PubMed
1. Henkel A. W., Meiri H., Horstmann H., Lindau M., Almers W. (2000). Rhythmic opening and closing of vesicles during constitutive exo- and endocytosis in chromaffin cells. EMBO J. 19 84–93. 10.1093/emboj/19.1.84 - DOI - PMC - PubMed
1. Henkel A. W., Welzel O., Groemer T. W., Tripal P., Rotter A., Kornhuber J. (2010). Fluoxetine prevents stimulation-dependent fatigue of synaptic vesicle exocytosis in hippocampal neurons. J. Neuro. 114 697–705. 10.1111/j.1471-4159.2010.06795.x - DOI - PubMed
1. Heuser J. E., Reese T. S. (1973). Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57 315–344. 10.1083/jcb.57.2.315 - DOI - PMC - PubMed
1. Jonas P., Bischofberger J., Sandkuhler J. (1998). Corelease of two fast neurotransmitters at a central synapse. Science 281 419–424. 10.1126/science.281.5375.419 - DOI - PubMed
1. Jordan R., Lemke E. A., Klingauf J. (2005). Visualization of synaptic vesicle movement in intact synaptic boutons using fluorescence fluctuation spectroscopy. Biophys. J. 89 2091–2102. 10.1529/biophysj.105.061663 - DOI - PMC - PubMed
1. Jung J., Loy K., Schilling E. M., Röther M., Brauner J. M., Huth T., et al. (2014). The antidepressant fluoxetine mobilizes vesicles to the recycling pool of rat hippocampal synapses during high activity. Mol. Neuro. 49 916–930. 10.1007/s12035-013-8569-5 - DOI - PubMed
1. Kim H. J., Kim T. H., Choi S. J., Hong Y. J., Yang J. S., Sung K. W., et al. (2013). Fluoxetine suppresses synaptically induced [Ca(2)(+)]i spikes and excitotoxicity in cultured rat hippocampal neurons. Brain Res. 1490 23–34. 10.1016/j.brainres.2012.10.062 - DOI - PubMed
1. Klingauf J., Kavalali E. T., Tsien R. W. (1998). Kinetics and regulation of fast endocytosis at hippocampal synapses. Nature 394 581–585. 10.1038/29079 - DOI - PubMed
1. Kraszewski K., Daniell L., Mundigl O., De Camilli P. (1996). Mobility of synaptic vesicles in nerve endings monitored by recovery from photobleaching of synaptic vesicle-associated fluorescence. J. Neurosci. 16 5905–5913. 10.1523/jneurosci.16-19-05905.1996 - DOI - PMC - PubMed
1. Megias M., Emri Z., Freund T. F., Gulyas A. I. (2001). Total number and distribution of inhibitory and excitatory synapses on hippocampal CA1 pyramidal cells. Neuroscience 102 527–540. 10.1016/s0306-4522(00)00496-6 - DOI - PubMed
1. Mouihate A., Kalakh S., AlMutairi R., Alashqar A. (2019). Prenatal inflammation dampens neurogenesis and enhances serotonin transporter expression in the hippocampus of adult female rats. Med. Princ. Pract. 28 352–360. 10.1159/000499658 - DOI - PMC - PubMed
1. Neher E., Sakaba T. (2008). Multiple roles of calcium ions in the regulation of neurotransmitter release. Neuron 59 861–872. 10.1016/j.neuron.2008.08.019 - DOI - PubMed
1. Nusser Z. (2018). Creating diverse synapses from the same molecules. Curr. Opin. Neurobiol. 51 8–15. 10.1016/j.conb.2018.01.001 - DOI - PubMed
1. Petrov A. M., Zakyrjanova G. F., Yakovleva A. A., Zefirov A. L. (2015). Inhibition of protein kinase C affects on mode of synaptic vesicle exocytosis due to cholesterol depletion. Biochem. Biophys. Res. Commun. 456 145–150. 10.1016/j.bbrc.2014.11.049 - DOI - PubMed
1. Qin X., Tsien R. W., Park H. (2019). Real-time three-dimensional tracking of single synaptic vesicles reveals that synaptic vesicles undergoing kiss-and-run fusion remain close to their original fusion site before reuse. Biochem. Biophys. Res. Commun. 514 1004–1008. 10.1016/j.bbrc.2019.05.043 - DOI - PubMed
1. Rao T. C., Passmore D. R., Peleman A. R., Das M., Chapman E. R., Anantharam A. (2014). Distinct fusion properties of synaptotagmin-1 and synaptotagmin-7 bearing dense core granules. Mol. Biol. Cell. 25 2416–2427. 10.1091/mbc.E14-02-0702 - DOI - PMC - PubMed
1. Ratnayaka A., Marra V., Bush D., Burden J. J., Branco T., Staras K. (2012). Recruitment of resting vesicles into recycling pools supports NMDA receptor-dependent synaptic potentiation in cultured hippocampal neurons. J. Physiol. 590 1585–1597. 10.1113/jphysiol.2011.226688 - DOI - PMC - PubMed
1. Richards D. A., Bai J., Chapman E. R. (2005). Two modes of exocytosis at hippocampal synapses revealed by rate of FM1-43 efflux from individual vesicles. J. Cell. Biol. 168 929–939. 10.1083/jcb.200407148 - DOI - PMC - PubMed
1. Rudling J. E., Drever B. D., Reid B., Bewick G. S. (2018). Importance of full-collapse vesicle exocytosis for synaptic fatigue-resistance at rat fast and slow muscle neuromuscular junctions. Int. J. Mol. Sci. 19:E1936. 10.3390/ijms19071936 - DOI - PMC - PubMed
1. Sakaba T. (2008). Two Ca(2+)-dependent steps controlling synaptic vesicle fusion and replenishment at the cerebellar basket cell terminal. Neuron 57 406–419. 10.1016/j.neuron.2007.11.029 - DOI - PubMed
1. Sankaranarayanan S., De Angelis D., Rothman J. E., Ryan T. A. (2000). The use of pHluorins for optical measurements of presynaptic activity. Biophys. J. 79 2199–2208. 10.1016/s0006-3495(00)76468-x - DOI - PMC - PubMed
1. Sankaranarayanan S., Ryan T. A. (2000). Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system. Nat. Cell. Biol. 2 197–204. 10.1038/35008615 - DOI - PubMed
1. Scepek S., Coorssen J. R., Lindau M. (1998). Fusion pore expansion in horse eosinophils is modulated by Ca2+ and protein kinase C via distinct mechanisms. EMBO J. 17 4340–4345. 10.1093/emboj/17.15.4340 - DOI - PMC - PubMed
1. Schikorski T., Stevens C. F. (2001). Morphological correlates of functionally defined synaptic vesicle populations. Nat. Neurosci. 4 391–395. 10.1038/86042 - DOI - PubMed
1. Somogyi P., Katona L., Klausberger T., Lasztoczi B., Viney T. J. (2014). Temporal redistribution of inhibition over neuronal subcellular domains underlies state-dependent rhythmic change of excitability in the hippocampus. Philos Trans. R. Soc. Lond. B Biol. Sci. 369:20120518. 10.1098/rstb.2012.0518 - DOI - PMC - PubMed
1. Threadgill R., Bobb K., Ghosh A. (1997). Regulation of dendritic growth and remodeling by Rho. Rac, and Cdc42. Neuron 19 625–634. 10.1016/s0896-6273(00)80376-1 - DOI - PubMed
1. Welzel O., Henkel A. W., Stroebel A. M., Jung J., Tischbirek C. H., Ebert K., et al. (2011). Systematic heterogeneity of fractional vesicle pool sizes and release rates of hippocampal synapses. Biophys. J. 100 593–601. 10.1016/j.bpj.2010.12.3706 - DOI - PMC - PubMed
1. Welzel O., Tischbirek C. H., Jung J., Kohler E. M., Svetlitchny A., Henkel A. W., et al. (2010). Synapse clusters are preferentially formed by synapses with large recycling pool sizes. PLoS One 5:e13514. 10.1371/journal.pone.0013514 - DOI - PMC - PubMed
1. Wu W., Xu J., Wu X. S., Wu L. G. (2005). Activity-dependent acceleration of endocytosis at a central synapse. J. Neurosci. 25 11676–11683. 10.1523/jneurosci.2972-05.2005 - DOI - PMC - PubMed
1. Wu Z., Auclair S. M., Bello O., Vennekate W., Dudzinski N. R., Krishnakumar S. S., et al. (2016). Nanodisc-cell fusion: control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains. Sci. Rep. 6 27287. 10.1038/srep27287 - DOI - PMC - PubMed
1. Xia X., Lessmann V., Martin T. F. (2009). Imaging of evoked dense-core-vesicle exocytosis in hippocampal neurons reveals long latencies and kiss-and-run fusion events. J. Cell Sci. 122 75–82. 10.1242/jcs.034603 - DOI - PMC - PubMed
1. Yan W., Liu J. F., Han Y., Zhang W., Luo Y. X., Xue Y. X., et al. (2017). Protein kinase Mzeta in medial prefrontal cortex mediates depressive-like behavior and antidepressant response. Mol. Psychiatry 23 1878–1891. 10.1038/mp.2017.219 - DOI - PubMed
1. Yi J. H., Zhang J., Ko S. Y., Kwon H., Jeon S. J., Park S. J., et al. (2018). Fluoxetine inhibits natural decay of long-term memory via Akt/GSK-3beta signaling. Mol. Neurobiol. 55 7453–7462. 10.1007/s12035-018-0919-x - DOI - PubMed
1. Zhang Q., Li Y., Tsien R. W. (2009). The dynamic control of kiss-and-run and vesicular reuse probed with single nanoparticles. Science 323 1448–1453. 10.1126/science.1167373 - DOI - PMC - PubMed

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