A Toolkit for Mapping and Modulating Neurotransmission at Single-Cell Resolution.

Understanding the organization and regulation of neurotransmission at the level of individual neurons and synapses requires tools that can track and manipulate transmitter-specific vesicles . Here, we present a suite of genetic tools in to fluorescently label and conditionally ablate the vesicular transporters for glutamate, GABA, acetylcholine, and monoamines. Using a structure-guided approach informed by protein topology and evolutionary conservation, we engineered endogenously tagged versions for each transporter that maintain their physiological function while allowing for cell-specific, bright, and stable visualization. We also developed conditional knockout strains that enable targeted disruption of neurotransmitter synthesis or packaging in single neurons. We applied this toolkit to map co-expression of vesicular transporters across the nervous system, revealing that over 10% of neurons exhibit co-transmission. Using the ADF sensory neuron as a case study, we demonstrate that serotonin and acetylcholine are trafficked in partially distinct vesicle pools. Our approach provides a powerful platform for mapping, monitoring, and manipulating neurotransmitter identity and use . The molecular strategies described here are likely applicable across species, offering a generalizable approach to dissect synaptic communication .