Technology provides means for studying neural circuits

In a new study in Neuron, they used trans-Tango to illuminate connected neurons in fruit flies, revealing previously unmapped gustatory circuits that link the taste-sensing organs to brain regions known to govern feeding behaviour and memory.

The technology is widely applicable, the researchers say, because trans-Tango doesn’t depend on the neurotransmitters involved in a neural connection or on the types of neurons that are connected. As long as two neurons join at a synapse, trans-Tango allows scientists to label the cells connected to a starter neuron, experiments in the paper show.

Moreover, because trans-Tango works by instigating the expression of genes in connected pairs of neurons, it also has the potential to enable scientists to control circuit functions, said senior and corresponding author Gilad Barnea, an associate professor of neuroscience at Brown who began looking for a precise, reliable and general way to visualise neural connections two decades ago.

The application of trans-Tango that his team demonstrates in the new study is circuit tracing, but manipulations such as activating or shutting off connected neurons could become possible, too.

“trans-Tango provides genetic accessibility in the context of connectivity,” Barnea said. “Our technique allows you to access the neurons that interact with the particular ‘starter’ cell you target. It therefore expands the use of molecular genetic techniques beyond the cell for which you have a marker to the ones it ‘talks’ to.”

The team, which includes postdoctoral fellows, graduate students, research assistants and undergraduates, is now working on developing a host of other applications of trans-Tango. These include using the system to manipulate behaviour, developing the equivalent technique in mice, and making it work in reverse so that it employs incoming connections from other neurons just like it does outgoing connections.

That’s according to Mustafa Talay, a postdoctoral fellow who earned his Ph.D. in Barnea’s lab and is co-lead author with Ethan Richman, a former undergraduate at Brown who is now a graduate student at Stanford. In addition, the Barnea lab is collaborating on adapting the technology to study how cancer spreads.

trans-Tango works by genetically introducing an artificial signaling pathway into every neuron in the fly. The pathway acts like a switch in the neurons that can be thrown by exposure to a triggering protein.

To operate trans-Tango, scientists genetically engineer the neurons of interest (starter neurons) to present this triggering protein on their synapses together with a protein that lights up the starter neurons in green.

Expression of the trigger protein at the synapse causes connected neurons to light up in red, revealing the full extent of the connected neurons in the fly’s nervous system.

In the gustatory system, for example, the team lit up connections extending all the way from peripheral taste-sensing starter neurons to connected neurons that projected into a brain region known to control feeding behaviour as well as to other regions thought to regulate memory.

By design, the system stops after just one stage of connectivity because if it continued endlessly, it would eventually light up the whole nervous system, Talay said. After all, each neuron usually connects to many others, not just one or a few, and ultimately they are pretty much all connected.

But the system is compatible with other cell imaging and targeting methods that can narrow down the number of connected neurons that respond to trans-Tango. In the new study, for example, the team combined trans-Tango with such techniques to specifically highlight individual connected neurons.

“When we probe a circuit we have no idea about, we can first just use trans-Tango and see the totality of all the connections of a neuron,” Talay said. “After that, if we want to characterise a circuit in more detail, we can combine trans-Tango with other methods to basically dissect that circuit.”

In many cases, revealing the full expanse that two connected neurons cover in a circuit can present deeply meaningful insights for neuroscientists. Not only did the team find novel connections in the gustatory circuitry of flies, but also they showed the different projections that various neurons in the olfactory system make, potentially clarifying how they carry out their distinct roles in connecting smell and behaviour.

Their experiments also highlighted connections that were already well known in the olfactory system, validating that the connections trans-Tango highlights are real.

The technology’s triggering protein is not naturally found in the fly, and it doesn’t leave the neurons or the synapse. For this reason, the scientists said, the illumination that arises as a result of trans-Tango reveals cells that truly “talk” to each other rather than neighboring but irrelevant cells.