Neuroscientists still possess an incomplete understanding of how different neurons interface and communicate throughout the brain’s wiring diagram, called the connectome. In a recent eLife article, Caltech molecular neurobiologist Carlos Lois and colleagues introduce a new tool to screen for such connections. The central question: When a neuron sends an electric signal, or action potential, down its axon, which neurons’ dendrites are directly downstream, listening for that signal?

Seeking answers, the authors built a three-part system centered in the synapse itself, where axons and dendrites meet. The method, called TRACT for TRAnsneuronal Control of Transcription, identifies interactions between an upstream neuron or neural population that’s chosen by the researchers, and unknown downstream neurons.

The researchers placed the first part of the system in the presynaptic terminals of select neurons of Drosophila, which would serve as the upstream neurons in the sought-after interactions. This part consists of the membrane and extracellular portions of any protein not normally found in brain cells—in this instance, the researchers used CD19 normally found on white blood cells.

The neurons contacted by this population could be anywhere in the brain. So the second part of TRACT is a hybrid protein made by all brain neurons, localized to postsynaptic terminals. Outside the cell, it contains an antibody chain that recognizes and binds CD19. Inside, it has a transcription factor called Gal4, tethered to the membrane. When CD19 from the upstream cell binds the antibody, this sets in motion a chain of events in which the protease gamma-secretase, already present in neurons, liberates Gal4 from the hybrid protein.

The transcription factor goes to the nucleus and turns on the gene for the third part of the system, green fluorescent protein. In that way, any neuron touching the upstream cell at a synapse will glow green.

To test their system, the authors chose Drosophila’s well-documented olfactory system, and confirmed that when they expressed the CD19 construct in olfactory neurons, TRACT lit up the neurons known to listen out downstream.

“I’m always happy to see new methods,” says Amita Sehgal, a neuroscientist at the Perelman School of Medicine of the University of Pennsylvania in Philadelphia, who was not involved with the study. But Sehgal sees the potential for false negatives with TRACT. In their eLife paper, Lois and colleagues also tested it out in the fly circadian rhythm circuit, which Sehgal studies. TRACT didn’t detect some neural connections that, based on her own and others’ work, Sehgal is confident do exist. That might be because the antibody-Gal4 hybrid was not expressed in all potential downstream neurons, she suggests.

“No technique is perfect,” says Lois. He and his colleagues hypothesize that false negatives might happen when there are very few connections between a pair of neurons. False positives might occur between neurons that are physically near each other but not directly connected by synapses.

Lois isn’t deterred—right now, he sees TRACT as a screening method that can identify potential neural partners. Other methods, such as electron microscopy (EM), would be required to confirm any links suggested by TRACT. But unlike EM, which is commonly used in connectomics, researchers can use TRACT to not only focus on specific populations of upstream neurons, but also highlight long-range connections. TRACT also works in live animals, and is less labor-intensive than EM, so that scientists could in principle assess several animals, says Lois.

“We could use it to detect how the wiring diagram changes in [animals with] different mutations,” he says, noting that conditions such as autism and schizophrenia are thought to stem from altered connectomes. Lois is currently adapting TRACT to work in mouse models.

TRACT joins another method reported last month, called trans-Tango, that traces neural circuits across the synapse in a similar manner. Like TRACT, trans-Tango depends on the release of a membrane-bound transcription factor when that interaction occurs. But while TRACT uses a protease already found in the cell, gamma-secretase, to release the transcription factor, trans-Tango uses the viral protease TEV, provided as part of the artificial system. The developers of trans-Tango also tested their system in olfactory neurons, and saw more connections than expected in some cases, suggesting possible false positives. Lois and colleagues suggest that further research will optimize both systems, as well as determine the rate of false results.