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

Nature Neuroscience volume 19, pages 578586 (2016) | Download Citation

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.

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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

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

    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.).

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

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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.

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.

Corresponding authors

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

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DOI

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

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