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