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pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity

Nature Neuroscience volume 15, pages 1047–1053 (2012)Cite this article

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Abstract

The usefulness of genetically encoded probes for optical monitoring of neuronal activity and brain circuits would be greatly advanced by the generation of multiple indicators with non-overlapping color spectra. Most existing indicators are derived from or spectrally convergent on GFP. We generated a bright, red, pH-sensitive fluorescent protein, pHTomato, that can be used in parallel with green probes to monitor neuronal activity. SypHTomato, made by fusing pHTomato to the vesicular membrane protein synaptophysin, reported activity-dependent exocytosis as efficiently as green reporters. When expressed with the GFP-based indicator GCaMP3 in the same neuron, sypHTomato enabled concomitant imaging of transmitter release and presynaptic Ca2+ transients at single nerve terminals. Expressing sypHTomato and GCaMP3 in separate cells enabled the simultaneous determination of presynaptic vesicular turnover and postsynaptic sub- and supra-threshold responses from a connected pair of neurons. With these new tools, we observed a close size matching between pre- and postsynaptic compartments, as well as interesting target cell–dependent regulation of presynaptic vesicle pools. Lastly, by coupling expression of pHTomato- and GFP-based probes with distinct variants of channelrhodopsin, we provided proof-of-principle for an all-optical approach to multiplex control and tracking of distinct circuit pathways.

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Figure 1: pHTomato spectra and properties.
Figure 2: Dual-color simultaneous imaging of vesicle fusion and cytosolic Ca2+.
Figure 3: Dual-color imaging of synaptic connection by sypHTomato and GCaMP3.
Figure 4: Dynamic imaging of synaptic transmission in suprathreshold or subthreshold condition by presynaptic sypHTomato and postsynaptic GCaMP3.
Figure 5: Proof of principle for an all-optical control and readout system.

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Acknowledgements

We thank M. Lin for help with quantum yield measurements. We thank J. Emery for technical assistance and L. Li for help with transfections. We thank L. Luo and Tsien laboratory members, especially M. Tadross, S. Owen, H. Park and R. Groth, for discussions throughout the execution of this project. This work was supported by grants from the US National Institute of Neurological Disorders and Stroke (NS074785 and NS24067), the US National Institute of Mental Health (MH064070), the Mathers Foundation and the Burnett family fund (to R.W.T.).

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Authors and Affiliations

  1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, USA

    Yulong Li & Richard W Tsien

  2. NYU Neuroscience Institute, New York University, New York, New York, USA

    Richard W Tsien

  3. Department of Physiology and Neuroscience, New York University, New York, New York, USA

    Richard W Tsien

  4. Center for Neural Science, New York University, New York, New York, USA

    Richard W Tsien

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  1. Yulong Li
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Y.L. conceived and performed the experiments. R.W.T. supervised the project. Y.L. and R.W.T. wrote the manuscript.

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Correspondence to Yulong Li or Richard W Tsien.

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Li, Y., Tsien, R. pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity. Nat Neurosci 15, 1047–1053 (2012). https://doi.org/10.1038/nn.3126

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