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

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• Genetically encoded fluorescent probes are crucial for optical monitoring of neuronal activity.
• Existing probes often share overlapping spectra, limiting multiplexing capabilities.
• Development of spectrally distinct probes is needed to advance brain circuit analysis.

Purpose of the Study:

• To generate novel fluorescent probes with non-overlapping spectra for enhanced neuronal monitoring.
• To develop a red fluorescent protein probe for parallel use with green probes.
• To enable simultaneous optical measurement of neuronal activity and neurotransmitter release.

Main Methods:

• Generation of a bright, red, pH-sensitive fluorescent protein (pHTomato).
• Fusion of pHTomato to synaptophysin to create SypHTomato for reporting exocytosis.
• Co-expression of SypHTomato with GFP-based indicators (e.g., GCaMP3) in neurons.
• Coupling probe expression with channelrhodopsin variants for all-optical control.

Main Results:

• pHTomato demonstrated utility as a red fluorescent probe for neuronal activity.
• SypHTomato efficiently reported activity-dependent exocytosis, comparable to green reporters.
• Simultaneous imaging of transmitter release and presynaptic Ca(2+) transients was achieved.
• All-optical multiplex control and tracking of distinct circuit pathways were demonstrated.

Conclusions:

• The development of pHTomato and SypHTomato expands the toolkit for multicolor optical monitoring of neural circuits.
• These probes facilitate simultaneous measurement of pre- and postsynaptic activity with high spatiotemporal resolution.
• The findings provide a foundation for advanced all-optical interrogation of complex neural pathways.