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

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 Ca(2+) 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.