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

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 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.

SUBMITTER: Li Y 

PROVIDER: S-EPMC3959862 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature