Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Fluorescent false neurotransmitter reveals functionally silent dopamine vesicle clusters in the striatum

Nature Neuroscience volume 19, pages 578–586 (2016)Cite this article

Subjects

Abstract

Neurotransmission at dopaminergic synapses has been studied with techniques that provide high temporal resolution, but cannot resolve individual synapses. To elucidate the spatial dynamics and heterogeneity of individual dopamine boutons, we developed fluorescent false neurotransmitter 200 (FFN200), a vesicular monoamine transporter 2 (VMAT2) substrate that selectively traces monoamine exocytosis in both neuronal cell culture and brain tissue. By monitoring electrically evoked Ca2+ transients with GCaMP3 and FFN200 release simultaneously, we found that only a small fraction of dopamine boutons that exhibited Ca2+ influx engaged in exocytosis, a result confirmed with activity-dependent loading of the endocytic probe FM1-43. Thus, only a low fraction of striatal dopamine axonal sites with uptake-competent VMAT2 vesicles are capable of transmitter release. This is consistent with the presence of functionally 'silent' dopamine vesicle clusters and represents, to the best of our knowledge, the first report suggestive of presynaptically silent neuromodulatory synapses.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure, spectral properties and selectivity of FFN200.
Figure 2: Effect of DAT and VMAT2 inhibition or genetic ablation on FFN200 loading in the dorsal striatum.
Figure 3: KCl-induced destaining of FFN200 in striatal slices and ventral midbrain cell cultures.
Figure 4: Electrically evoked FFN200 release in striatal slices.
Figure 5: FFN200 release dependence on stimulus frequency in dorsal striatum.
Figure 6: Identification of active presynaptic dopaminergic boutons in the dorsal striatum by FM1-43 loading.
Figure 7: Quasi-simultaneous imaging of FFN200 destaining and GCaMP3-monitored Ca2+ transients in striatal dopamine axonal sites.

Similar content being viewed by others

References

  1. Zhang, M.Y. & Beyer, C.E. Measurement of neurotransmitters from extracellular fluid in brain by in vivo microdialysis and chromatography-mass spectrometry. J. Pharm. Biomed. Anal. 40, 492–499 (2006).

    CAS  PubMed  Google Scholar 

  2. Schmitz, Y., Lee, C.J., Schmauss, C., Gonon, F. & Sulzer, D. Amphetamine distorts stimulation-dependent dopamine overflow: effects on D2 autoreceptors, transporters, and synaptic vesicle stores. J. Neurosci. 21, 5916–5924 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Kennedy, R.T., Jones, S.R. & Wightman, R.M. Simultaneous measurement of oxygen and dopamine: coupling of oxygen consumption and neurotransmission. Neuroscience 47, 603–612 (1992).

    CAS  PubMed  Google Scholar 

  4. Bérubé-Carrière, N. et al. Ultrastructural characterization of the mesostriatal dopamine innervation in mice, including two mouse lines of conditional VGLUT2 knockout in dopamine neurons. Eur. J. Neurosci. 35, 527–538 (2012).

    PubMed  Google Scholar 

  5. Pickel, V.M., Beckley, S.C., Joh, T.H. & Reis, D.J. Ultrastructural immunocytochemical localization of tyrosine hydroxylase in the neostriatum. Brain Res. 225, 373–385 (1981).

    CAS  PubMed  Google Scholar 

  6. Betz, W.J. & Bewick, G.S. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science 255, 200–203 (1992).

    CAS  PubMed  Google Scholar 

  7. Miesenböck, G., De Angelis, D.A. & Rothman, J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    PubMed  Google Scholar 

  8. Onoa, B., Li, H., Gagnon-Bartsch, J.A., Elias, L.A. & Edwards, R.H. Vesicular monoamine and glutamate transporters select distinct synaptic vesicle recycling pathways. J. Neurosci. 30, 7917–7927 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Mani, M. & Ryan, T.A. Live imaging of synaptic vesicle release and retrieval in dopaminergic neurons. Front. Neural Circuits 3, 3 (2009).

    PubMed  PubMed Central  Google Scholar 

  10. Pan, P.Y. & Ryan, T.A. Calbindin controls release probability in ventral tegmental area dopamine neurons. Nat. Neurosci. 15, 813–815 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Daniel, J.A., Galbraith, S., Iacovitti, L., Abdipranoto, A. & Vissel, B. Functional heterogeneity at dopamine release sites. J. Neurosci. 29, 14670–14680 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Gubernator, N.G. et al. Fluorescent false neurotransmitters visualize dopamine release from individual presynaptic terminals. Science 324, 1441–1444 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Rodriguez, P.C. et al. Fluorescent dopamine tracer resolves individual dopaminergic synapses and their activity in the brain. Proc. Natl. Acad. Sci. USA 110, 870–875 (2013).

    CAS  PubMed  Google Scholar 

  14. Liu, Y. et al. A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter. Cell 70, 539–551 (1992).

    CAS  PubMed  Google Scholar 

  15. Matsushita, N. et al. Dynamics of tyrosine hydroxylase promoter activity during midbrain dopaminergic neuron development. J. Neurochem. 82, 295–304 (2002).

    CAS  PubMed  Google Scholar 

  16. Fuxe, K., Hökfelt, T., Olson, L. & Ungerstedt, U. Central monoaminergic pathways with emphasis on their relation to the so called 'extrapyramidal motor system'. Pharmacol. Ther. [B] 3, 169–210 (1977).

    CAS  Google Scholar 

  17. Pickel, V.M., Nirenberg, M.J. & Milner, T.A. Ultrastructural view of central catecholaminergic transmission: immunocytochemical localization of synthesizing enzymes, transporters and receptors. J. Neurocytol. 25, 843–856 (1996).

    CAS  PubMed  Google Scholar 

  18. Thompson, L., Barraud, P., Andersson, E., Kirik, D. & Björklund, A. Identification of dopaminergic neurons of nigral and ventral tegmental area subtypes in grafts of fetal ventral mesencephalon based on cell morphology, protein expression, and efferent projections. J. Neurosci. 25, 6467–6477 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Caudle, W.M. et al. Reduced vesicular storage of dopamine causes progressive nigrostriatal neurodegeneration. J. Neurosci. 27, 8138–8148 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Mooslehner, K.A. et al. Mice with very low expression of the vesicular monoamine transporter 2 gene survive into adulthood: potential mouse model for parkinsonism. Mol. Cell. Biol. 21, 5321–5331 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Grace, A.A. & Bunney, B.S. The control of firing pattern in nigral dopamine neurons: burst firing. J. Neurosci. 4, 2877–2890 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Kennedy, R.T., Jones, S.R. & Wightman, R.M. Dynamic observation of dopamine autoreceptor effects in rat striatal slices. J. Neurochem. 59, 449–455 (1992).

    CAS  PubMed  Google Scholar 

  23. Abeliovich, A. et al. Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system. Neuron 25, 239–252 (2000).

    CAS  PubMed  Google Scholar 

  24. Arbuthnott, G.W. & Wickens, J. Space, time and dopamine. Trends Neurosci. 30, 62–69 (2007).

    CAS  PubMed  Google Scholar 

  25. Groves, P.M., Linder, J.C. & Young, S.J. 5-hydroxydopamine-labeled dopaminergic axons: three-dimensional reconstructions of axons, synapses and postsynaptic targets in rat neostriatum. Neuroscience 58, 593–604 (1994).

    CAS  PubMed  Google Scholar 

  26. Altrock, W.D. et al. Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon. Neuron 37, 787–800 (2003).

    CAS  PubMed  Google Scholar 

  27. Rosenmund, C. et al. Differential control of vesicle priming and short-term plasticity by Munc13 isoforms. Neuron 33, 411–424 (2002).

    CAS  PubMed  Google Scholar 

  28. Moulder, K.L. et al. Plastic elimination of functional glutamate release sites by depolarization. Neuron 42, 423–435 (2004).

    CAS  PubMed  Google Scholar 

  29. Kannenberg, K., Sieghart, W. & Reuter, H. Clusters of GABAA receptors on cultured hippocampal cells correlate only partially with functional synapses. Eur. J. Neurosci. 11, 1256–1264 (1999).

    CAS  PubMed  Google Scholar 

  30. Cousin, M.A. & Evans, G.J. Activation of silent and weak synapses by cAMP-dependent protein kinase in cultured cerebellar granule neurons. J. Physiol. (Lond.) 589, 1943–1955 (2011).

    CAS  Google Scholar 

  31. Sgobio, C. et al. Optogenetic measurement of presynaptic calcium transients using conditional genetically encoded calcium indicator expression in dopaminergic neurons. PLoS One 9, e111749 (2014).

    PubMed  PubMed Central  Google Scholar 

  32. Zariwala, H.A. et al. A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo. J. Neurosci. 32, 3131–3141 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Bäckman, C.M. et al. Characterization of a mouse strain expressing Cre recombinase from the 3′ untranslated region of the dopamine transporter locus. Genesis 44, 383–390 (2006).

    PubMed  Google Scholar 

  34. Zakharenko, S.S., Zablow, L. & Siegelbaum, S.A. Visualization of changes in presynaptic function during long-term synaptic plasticity. Nat. Neurosci. 4, 711–717 (2001).

    CAS  PubMed  Google Scholar 

  35. Zhang, H. & Sulzer, D. Frequency-dependent modulation of dopamine release by nicotine. Nat. Neurosci. 7, 581–582 (2004).

    CAS  PubMed  Google Scholar 

  36. Rice, M.E. & Cragg, S.J. Nicotine amplifies reward-related dopamine signals in striatum. Nat. Neurosci. 7, 583–584 (2004).

    CAS  PubMed  Google Scholar 

  37. Jahromi, S.S. & Atwood, H.L. Three-dimensional ultrastructure of the crayfish neuromuscular apparatus. J. Cell Biol. 63, 599–613 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Hirst, G.D., Redman, S.J. & Wong, K. Post-tetanic potentiation and facilitation of synaptic potentials evoked in cat spinal motoneurones. J. Physiol. (Lond.) 321, 97–109 (1981).

    CAS  Google Scholar 

  39. Bennett, M., Jones, P. & Lavidis, N. Transmitter secretion varies between visualized release sites at amphibian neuromuscular junctions. Neurosci. Lett. 65, 311–315 (1986).

    CAS  PubMed  Google Scholar 

  40. Losonczy, A., Biró, A.A. & Nusser, Z. Persistently active cannabinoid receptors mute a subpopulation of hippocampal interneurons. Proc. Natl. Acad. Sci. USA 101, 1362–1367 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Chavis, P., Mollard, P., Bockaert, J. & Manzoni, O. Visualization of cyclic AMP-regulated presynaptic activity at cerebellar granule cells. Neuron 20, 773–781 (1998).

    CAS  PubMed  Google Scholar 

  42. Moulder, K.L., Jiang, X., Taylor, A.A., Olney, J.W. & Mennerick, S. Physiological activity depresses synaptic function through an effect on vesicle priming. J. Neurosci. 26, 6618–6626 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Jiang, X. et al. A role for the ubiquitin-proteasome system in activity-dependent presynaptic silencing. J. Neurosci. 30, 1798–1809 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Matsuda, W. et al. Single nigrostriatal dopaminergic neurons form widely spread and highly dense axonal arborizations in the neostriatum. J. Neurosci. 29, 444–453 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Cacciapaglia, F., Wightman, R.M. & Carelli, R.M. Rapid dopamine signaling differentially modulates distinct microcircuits within the nucleus accumbens during sucrose-directed behavior. J. Neurosci. 31, 13860–13869 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Robinson, D.L., Howard, E.C., McConnell, S., Gonzales, R.A. & Wightman, R.M. Disparity between tonic and phasic ethanol-induced dopamine increases in the nucleus accumbens of rats. Alcohol. Clin. Exp. Res. 33, 1187–1196 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Choi, S., Klingauf, J. & Tsien, R.W. Postfusional regulation of cleft glutamate concentration during LTP at 'silent synapses'. Nat. Neurosci. 3, 330–336 (2000).

    CAS  PubMed  Google Scholar 

  48. Ma, L., Zablow, L., Kandel, E.R. & Siegelbaum, S.A. Cyclic AMP induces functional presynaptic boutons in hippocampal CA3-CA1 neuronal cultures. Nat. Neurosci. 2, 24–30 (1999).

    CAS  PubMed  Google Scholar 

  49. Voronin, L.L. et al. Postsynaptic depolarisation enhances transmitter release and causes the appearance of responses at “silent” synapses in rat hippocampus. Neuroscience 126, 45–59 (2004).

    CAS  PubMed  Google Scholar 

  50. Porrill, J. & Dean, P. Silent synapses, LTP, and the indirect parallel-fibre pathway: computational consequences of optimal cerebellar noise-processing. PLoS Comput. Biol. 4, e1000085 (2008).

    PubMed  PubMed Central  Google Scholar 

  51. Adam, Y., Edwards, R.H. & Schuldiner, S. Expression and function of the rat vesicular monoamine transporter 2. Am. J. Physiol. Cell Physiol. 294, C1004–C1011 (2008).

    CAS  PubMed  Google Scholar 

  52. Giros, B., Jaber, M., Jones, S.R., Wightman, R.M. & Caron, M.G. Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 379, 606–612 (1996).

    CAS  PubMed  Google Scholar 

  53. Mena, M.A. et al. Effects of wild-type and mutated copper/zinc superoxide dismutase on neuronal survival and L-DOPA-induced toxicity in postnatal midbrain culture. J. Neurochem. 69, 21–33 (1997).

    CAS  PubMed  Google Scholar 

  54. Wong, M.Y., Sulzer, D. & Bamford, N.S. Imaging presynaptic exocytosis in corticostriatal slices. Methods Mol. Biol. 793, 363–376 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Zipfel, W.R., Williams, R.M. & Webb, W.W. Nonlinear magic: multiphoton microscopy in the biosciences. Nat. Biotechnol. 21, 1369–1377 (2003).

    CAS  PubMed  Google Scholar 

  57. Schmitz, Y., Schmauss, C. & Sulzer, D. Altered dopamine release and uptake kinetics in mice lacking D2 receptors. J. Neurosci. 22, 8002–8009 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank M. Caron (Duke University) for providing DAT knockout mice, A. Salahpour (University of Toronto) and G. Miller (Emory University) for the VMAT2 hypomorph, mice and K. Kobayashi (Fukushima Medical University) for the TH-GFP mice. We would also like to thank C. Castagna, V. Morales, A. Barnett and D. Korostyshevsky for excellent technical support as well as other members of the Sulzer and Sames laboratories for helpful discussions and support. This work was supported by the G. Harold & Leila Y. Mathers Charitable Foundation (D. Sames), National Institute on Mental Health (R01MH086545 to D. Sames, R01MH108186 to D. Sames and D. Sulzer), the J.P.B. (D. Sulzer), McKnight (D. Sames and D. Sulzer) and Parkinson's Disease Foundations (D. Sulzer), the National Institute on Drug Abuse (DA07418 and DA10154; D. Sulzer), the National Institute on Alcohol Abuse and Alcoholism (AA019801; D. Sulzer), and a National Institute of Neurological Disorders and Stroke (NINDS) Udall Center of Excellence for Parkinson's Disease Research (D. Sulzer). Y.S. was supported by the Michael J. Fox Foundation, J.E.L.-O. was supported by NINDS (3 P50 NS 038370-13S1), P.C.R. was granted a pre-doctoral fellowship by the National Science Foundation and E.V.M. was funded by NINDS (R01NS075222).

Author information

Author notes

  1. Daniela B Pereira, Gang Hu, Shu Li, Adam Henke, Richard J Karpowicz Jr & Pamela C Rodriguez

    Present address: Present addresses: Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Lisbon, Portugal (D.B.P.), Infineum USA, Linden, New Jersey, USA (G.H.), Pharmasan Labs, Osceola, Wisconsin, USA (S.L.), California Institute for Biomedical Research, La Jolla, California, USA (A.H.), Department of Pathology and Laboratory Medicine, Center for Neurodegenerative Disease Research, Perelman School of Medicine, University of Pennsylvania, Pennsylvania, USA (R.J.K.), Institute of Biology, École Normale Supérieure, Paris, France (P.C.R.).,

Authors and Affiliations

  1. Department of Neurology, Columbia University Medical Center, New York, New York, USA

    Daniela B Pereira, Yvonne Schmitz, José E Lizardi-Ortiz, Mark S Sonders, Ellen Kanter, Eugene V Mosharov & David Sulzer

  2. Department of Psychiatry, Columbia University Medical Center, New York, New York, USA

    József Mészáros, Mark S Sonders & David Sulzer

  3. Department of Chemistry, Columbia University, New York, New York, USA

    Paolomi Merchant, Gang Hu, Shu Li, Adam Henke, Richard J Karpowicz Jr, Pamela C Rodriguez & Dalibor Sames

  4. Department of Pharmacology, Columbia University Medical Center, New York, New York, USA

    Travis J Morgenstern & David Sulzer

  5. Department of Neuroscience, New York Psychiatric Institute, New York, New York, USA

    David Sulzer

Authors
  1. Daniela B Pereira
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yvonne Schmitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. József Mészáros
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Paolomi Merchant
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gang Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Adam Henke
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. José E Lizardi-Ortiz
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Richard J Karpowicz Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Travis J Morgenstern
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Mark S Sonders
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Ellen Kanter
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Pamela C Rodriguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Eugene V Mosharov
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Dalibor Sames
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. David Sulzer
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.B.P., Y.S., E.V.M., D. Sulzer and D. Sames conceived and designed the experiments. S.L. conducted the early examination of FFN200 in brain tissue. D.B.P., P.M. and P.C.R. performed slice imaging probe characterization experiments and Y.S. and E.V.M. performed cell culture imaging experiments. D.B.P. performed brain slice FFN200 destaining experiments, including FFN200-GCaMP3 simultaneous imaging. J.M., aided by D.B.P., developed the Matlab image analysis routine and T.J.M. contributed with additional data output scripts. D. Sames designed FFN200, and G.H. and A.H. designed and performed FFN200 synthesis and purification as well as chemical and photophysical characterization. R.J.K. performed the FFN200 Km determination experiments. J.E.L.-O. designed and performed the CV experiments. M.S.S. aided with the FFN200-GCaMP3 simultaneous imaging experiments. E.K. optimized and provided midbrain dopamine cultures. D.B.P., Y.S., P.M., J.E.L.-O., R.J.K., P.C.R. and E.V.M. analyzed data. D.B.P. wrote the paper, with important contributions from Y.S., J.M., M.S.S., E.V.M., P.C.R., D. Sulzer and D. Sames.

Corresponding authors

Correspondence to Daniela B Pereira, Dalibor Sames or David Sulzer.

Ethics declarations

Competing interests

D. Sulzer and D. Sames were listed as inventors on a patent (8,337,941) covering FFN200 and a patent application (13/575,535) covering FFN102, compounds employed in this study.

Integrated supplementary information

Supplementary Figure 1 FFN200 accumulates in cultured dopamine neurons.

(a) Representative images of FFN200-labeled dopamine ventral midbrain neurons obtained from TH-GFP mice, displaying different levels of FFN200 accumulation following incubation with 10 μM FFN200 for 30 min at 37°C. (b) Scatter plot of FFN200 intensity values at GFP-positive dopamine neurons, presented in ascending order in a logarithmic scale. Neurons were considered FFN200-positive when their fluorescence intensity was greater than two standard deviations above the mean FFN200 intensity of GFP-negative cells (the threshold is depicted as a dotted line; 80 cells from six dishes, four independent cultures). An example of an FFN200-negative neuron is shown in the top TH-GFP/FFN200 image pair in panel (a), and the following two image pairs show FFN200-positive neurons.

Supplementary Figure 2 Effect of dTBZ on dopamine release as a function of incubation time.

Single pulse-evoked dopamine release was measured by cyclic voltammetry in the dorsal striatum of control and 5 μM dTBZ-treated slices (n=3–4 mice with 1–2 slices averaged per mouse). Released dopamine was normalized for each condition to the average current of five prepulses applied before time point zero, which marks the beginning of dTBZ perfusion (in dTBZ-treated slices).

Supplementary Figure 3 Effect of 0 mM Ca2+ and TTX on FFN200 release in the dorsal striatum.

(a) Background intensity presented as mean percentage of Fi ± SEM for slices stimulated in the presence of 2.4 mM Ca2+, in the absence of Ca2+ (0 mM Ca2+) or in 2.4 mM Ca2+ with 1 μM TTX (2.4 mM Ca2+ + TTX; n=6 for each condition in both panels, where n is number of mice, with 1–2 slices averaged per mouse). (b) Scatter plot of the percentage of destaining puncta in each independent experiment including mean ± SEM (**p < 0.01 by one-way ANOVA with Bonferroni's multiple comparison test).

Supplementary Figure 4 Comparison of FFN200 and FFN102 release in the dorsal striatum.

(a) Background intensity presented as mean percentage of Fi ± SEM for both probes (n=9 and 8 slices from different mice for FFN200 and FFN102, respectively, for all panels in this figure). (b) Scatter plot of the percentage of FFN200 and FFN102 destaining puncta in response to 15 Hz stimuli in each independent experiment including mean ± SEM (n.s- not significantly different, p=0.1269, two-tailed unpaired t test). As a control, the percentage of FFN102 puncta selected as “destainers” in unstimulated slices was 5.1 ± 1.6% (n=5; not depicted). (c) FFN200 and FFN102 puncta intensity (background- and baseline rundown-corrected) over time, normalized to ΔF, the fluorescence change between the last point of the baseline (100%) and the average of the last three data points of stimulation (0%) ± SEM, to facilitate comparison between FFN200 and FFN102 curves (*p < 0.05, two-tailed unpaired t test; p=0.0370). (d) Cumulative distribution of t1/2s of destaining for FFN200 and FFN102 (147 and 175 puncta from nine and eight slices from different mice for FFN200 and FFN102, respectively).

Supplementary Figure 5 Effects of short washout times on FFN200 release in the dorsal striatum.

(a) Scatter plot of the percentage of destaining puncta in each independent experiment, including mean ± SEM, for typical 45 min washout (45’ washout) and shorter 25 min washout (25’ washout) (n=6 for both conditions in both panels, where n is number of mice, with 1–2 slices averaged per mouse; n.s- not significantly different, p=0.4771 by two-tailed unpaired t test). (b) Puncta fluorescence intensity (background- and baseline rundown-corrected) over time presented as mean percentage of Fi ± SEM.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pereira, D., Schmitz, Y., Mészáros, J. et al. Fluorescent false neurotransmitter reveals functionally silent dopamine vesicle clusters in the striatum. Nat Neurosci 19, 578–586 (2016). https://doi.org/10.1038/nn.4252

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.4252

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing